Fungus resistant plants and their uses

ABSTRACT

The present invention relates to a novel method for increasing the resistance, of a plant, in particular of a Solanaceae, preferably of potato and tomato, to plant pathogens of the phylum Oomyceta comprising increasing the activity of the polypeptid of the present invention. The invention further relates to polynucleotides and vectors comprising these polynucleotides. The invention furthermore relates to corresponding vectors, cells, transgenic plants and transgenic propagation material derived from them, methods to produce them and to their use for the production of foodstuffs, feeding stuffs, seed, pharmaceuticals or fine chemicals.

RELATED APPLICATIONS

This application is a national stage application (under 35 U.S.C. 371)of PCT/EP2004/008683 filed Aug. 3, 2004 which claims benefit of Europeanpatent application 03018266.1 filed Aug. 11, 2003.

SUBMISSION ON COMPACT DISC

The contents of the following submission on compact discs areincorporated herein by reference in its entirety: two copies of theSequence Listing (COPY 1 and COPY 2) and a computer readable form copyof the Sequence Listing (CRF COPY), all on compact disc , eachcontaining: file name: Sequence List-13477-00002-US, date recorded: Oct.24, 2007, size: 168 KB.

The present invention relates to a novel method for increasing theresistance of a plant, in particular of a Solanaceae, preferably ofpotato and tomato, to plant pathogens of the phylum Oomycetes comprisingincreasing the activity of the polypeptide of the present invention. Theinvention further relates to polynucleotides and vectors comprisingthese polynucleotides. The invention furthermore relates tocorresponding vectors, cells, transgenic plants and transgenicpropagation material derived from them, methods to produce them and totheir use for the production of foodstuffs, feeding stuffs, seed,pharmaceuticals or fine chemicals.

The aim of plant biotechnology work is the generation of plants withadvantageous novel properties, for example for increasing agriculturalproductivity, increasing the quality in the case of foodstuffs, or forproducing specific chemicals or pharmaceuticals (Dunwell J M (2000) JExp Bot 51 Spec No:487-96). The plant's natural defence mechanismsagainst pathogens are frequently insufficient. Fungal diseases aloneresult in annual yield losses of many billions of US$. The introductionof foreign genes from plants, animals or microbial sources can increasethe defences. Examples are the protection of tobacco against feedingdamage by insects by expressing Bacillus thuringiensis endotoxins underthe control of the 35S CaMV promoter (Vaeck et al. (1987) Nature328:33-37) or the protection of tobacco against fungal infection byexpressing a bean chitinase under the control of the CaMV promoter(Broglie et al. (1991) Science 254:1194-1197). However, most of theapproaches described only offer resistance to a single pathogen or anarrow spectrum of pathogens.

Despite the notorious Irish potato famine of the mid-19^(th) century,late blight still continues to be one of the most devastating of alldiseases in crop plants. Late blight is caused by the oomycete fungusPhytophthora infestans, a specialised pathogen, primarily causingdisease on the foliage and fruits of a range of Solanaceae species,especially potato and tomato. The fungus was first observed in Mexicoand for several reasons Mexico is believed to be the centre of origin ofthe fungus. Both of the mating types A1 and A2 are permanently presentin for example the Toluca area. Also, P. infestans is reported on nativeSolanum species in remote areas of Mexico. Furthermore, many species oftuber bearing Solanum with a high level of resistance to late blight arefound in Mexico. Prevailing measures to prevent crop failures or reducedyields imply the application of fungicides that prevent or cure aninfection by P. infestans. Instead of the massive use of chemicalpesticides an alternative approach for controlling late blight could beadvantageous: the use of cultivars, which harbour partial or completeresistance to late blight. To obtain late blight resistance, breedershave in the past focussed on the introgression of dominant R genes fromSolanum demissum, a wild potato species indigenous to Mexico. Elevensuch R genes have been identified, several of which have been mapped tospecific loci on the genetic map of potato (reviewed in Gebhardt andValkonen, 2001) and recently the R1 gene has been cloned. R1 and R2 arelocated on chromosomes 5 and 4, respectively. R3, R6 and R7 are locatedon chromosome 11. Unknown R genes conferring race specific resistance tolate blight have also been described in S. tuberosum ssp. andigena andS. berthaultii and S. pinnatisectum. The resistance induced by theseR-genes was (nearly) complete but appeared not to be durable in anycase. Because of the high level of resistance and ease of transfer, manycultivars contain S. demissum derived resistance. Unfortunately, S.demissum derived race specific resistance, although nearly complete, isnot durable. Once newly bred potato cultivars were grown on larger scalein commercial fields, new virulences emerged in P. infestans, whichrendered the pathogen able to overcome the introgressed resistance. Moredurable field resistance to late blight, often quantitative in natureand presumed to be race non-specific, can be found in several Mexicanand Central and South American Solanum species. However this type ofresistance is difficult to transfer into potato cultivars throughcrossing and phenotypic selection.

Diploid S. bulbocastanum from Mexico and Guatemala is one of the tuberbearing species that is long known for its high levels of resistance tolate blight. Unfortunately, classic transfer of resistance from wildSolanum species to cultivated potato is frequently prevented due todifferences in ploidy and Endosperm Balance Number (EBN). Despite theseproblems, introgression of the S. bulbocastanum resistance trait hasbeen successful. Recently, somatic hybrids of S. bulbocastanum and S.tuberosum and backcrossed germplasm were found to be highly resistant tolate blight, even under extreme disease pressure (Helgeson et al.,1998). Despite reports of suppression of recombination, resistance inthe backcrossed material appeared to be on chromosome 8 within anapproximately 6 cM interval between the RFLP markers CP53 and CT64. ACAPS marker derived from the tomato RFLP probe CT88 cosegregated withresistance.

Accordingly, in the recent years the development of plants resistant topathogens of the phylum Oomyceta forged ahead. However, 40 years ofintense and continuous research and breeding efforts with availablegermplasm has still not resulted in market introduction of resistantcultivars. The prevailing number of genes identified in the recent yearsconfers merely race specific resistance. Further, the achievedresistance was not durable. In addition, the application of cropprotectants is widely considered to be a burden for the environment.Thus, in several Western countries, legislation becomes more restrictiveand partly prohibitive to the application of specific fungicides, makingchemical control of the disease more difficult. Further, chemicalcontrol is expensive. Finally, another restriction is the development ofresistance by the fungus to specific fungicides such as metalaxyl, whichhas been reported from many countries in the world.

Accordingly, the problem underlying the present invention is to providenovel means and methods for an efficient protection of plants againstlate blight and related diseases.

The solution of the technical problem is achieved by providing theembodiments characterized in the claims.

Accordingly, the present invention relates to a method for generating orincreasing the resistance of a plant to plant pathogen of the phylumOomycetes comprising increasing the activity of Rpi-blb2 protein in theplant or a tissue, organ or cell of the plant or a part thereof.

Rpi-blb2 is a LZ-NBS-LRR type of R gene and shows sequence homology tothe tomato gene Mi-1, that confers resistance to three species of rootknot nematodes (Meloidogyne spp.) as well as to the potato aphidMacrosiphum euphorbiae (Vos et al., 1998; Rossi et al., 1998; Milliganet al., 1998) and to both B- and Q-biotypes of whitefly Bemisia tabaci(Nombela et al., 2003). As was found for Rpi-blb, Rpi-blb2 also confersfull resistance to a range of P. infestans isolates carrying multiplevirulence factors and race-specificity has not yet been demonstrated.

The term “Rpi-blb2” refers to a polynucleotide encoding a polypeptidehaving the herein mentioned Rpi-blb2 protein activity or a polypeptidehaving said Rpi-blb2 protein activity. Whether in the following the term“Rpi-blb2” relates to a polypeptide or a polynucleotide is clear fromthe context of its usage.

By the term “generating” or “increasing” or “stimulating” “theresistance of a plant” is meant that the resistance of a plant or a partthereof is increased or generated or stimulated in comparison to areference.

“Conferring”, “existing”, “generating”, “stimulating” or “increasing” apathogen resistance means that the defence mechanisms of a specificplant species or variety is increasingly resistant to one or morepathogens due to the use of the method according to the invention incomparison with the wild type of the plant, to which the methodaccording to the invention has not been applied, under otherwiseidentical conditions (such as, for example, climatic conditions, growingconditions, pathogen species and the like). The increased resistancemanifests itself preferably in a reduced manifestation of the diseasesymptoms, disease symptoms comprising—in addition to the abovementionedadverse effects—for example also the penetration efficiency of apathogen into the plant or plant cells or the proliferation efficiencyin or on the same. In this context, the disease symptoms are preferablyreduced by at least 10% or at least 20%, especially preferably by atleast 40% or 60%, very especially preferably by at least 70% or 80% andmost preferably by at least 90% or 95%.

By the term “increased” it is hereby meant that an activity of a geneproduct is higher than in a reference. Thus, the term “increased”includes that an activity, e.g. the activity of Rpi-blb2 gene product orof an other gene product, is generated de novo, if that activity, e.gthe herein described Rpi-blb2 activity, was not found in the reference.The term “increased” also relates to the stimulation of the activity ofa gene product. An increased expression of a gene, i.e. its activationcan be stimulated on several ways, e.g. by applying chemicals or bybiotic stress to an organism. For example, a resistance to infectingparasites mediating gene may be activated by infection with a parasite,e.g. with P. infestans and confers than an increased resistance to thesame and/or other pathogens.

Thus, in the following, the term “increasing” also comprises the terms“stimulating” and “generating”.

“Pathogen resistance” denotes the reduction or weakening of diseasesymptoms of a plant following infection by a pathogen. The symptoms canbe manifold, but preferably encompass those which directly or indirectlyhave an adverse effect on the quality of the plant, the quantity of theyield, the suitability for use as feeding stuff or foodstuff, or elsewhich make sowing, planting, harvesting or processing of the cropdifficult.

“Pathogen” within the scope of the invention means by way of example butnot by limitation viruses or viroids, bacteria, fungi, animal pests suchas, for example, insects or nematodes.

The term “Rpi-blb2 protein” relates to a protein or polypeptide whichexpression in a plant or a part confers resistance of the plant or apart of the plant to one of the pathogens described herein in comparisonto a non-resistant strain.

The plant or a tissue, organ or cell of the plant or a part thereofcomprising increased activity of Rpi-blb2 protein is less susceptible toan infection by a pathogen, in particular to pathogen of the phylumOomycetes, preferably to P. infestans, than a plant or a part thereofwhich has the identical genetic background but not the genetic elementsnecessary to allow an expression of Rpi-blb2 (herein named as “wildtype” or “reference”). Assays for the testing of the resistance of aplant or a part thereof are well known to a person skilled in the art.The resistance to P. infestans can be defined as sporulation indexaccording to Flier, 2001. Flier describes the sporulation index as alevel of sporulation per 1 cm². Thus, a reduction of sporulation per 1cm² of 20% compared to a wild type is herein defined as resistance. Inthe examples illustrating the present invention, the sporulation indexwas defined as level of sporulation per lesion. Thus, by the term“resistance” can be alternatively meant a reduction of sporulation perlesion of 20% compared to a wild type. The later definition ispreferred.

In preferred embodiments the sporulation in an assay is reduced by 30%,more preferred is a reduction of 50%, even more preferred are 70%, evenmore preferred are more than 80%, more preferred are 85% and 90%. Mostpreferred is a reduction of 95% or more.

Accordingly, in the present invention by “activity” of a Rpi-blb2protein is meant, that the protein expression confers said reduction inthe sporulation index. Further, it was observed, that a typical responsefor plants containing Rpi-blb2 to a P. infestans infection is thepresence of small lesions, without any clear sporulation, at the end ofthe growing season. Thus, in one embodiment, the activity of Rpi-blb2 isdefined as the presence of small lesions without any clear sporulationin experiments as described. Rpi-blb2 resistance shows necrotic regionsthat contain a low level of sporulation. An experiment performed withdetached leaves exemplifies the activity of Rpi-blb2. The experiment isdescribed in example 17 and FIG. 18. The difference between Rpi-blb2 andother P. infestans resistance genes is that Rpi-blb2 allows a low levelof sporulation (FIG. 18). A detached leaf assay in which the lesionspresent on Rpi-blb2 genotype (ARD 92-1197-16) shows a low level ofsporangia in relation to complete absence of sporangia on a genotypecontaining the S. demissum gene R2. The sporulation index is only 1.1%of a susceptible phenotype (cv. Bintje) (Table 7 and FIG. 18).

Field experiments have also shown that Rpi-blb2 allows a low level ofinfection. Late blight symptoms developed at a low level during thegrowing season (FIG. 3, ARF87-801) or at the end of the growing season(FIG. 2, ARF87-601; FIG. 3, ARF87-507 and ARF87-601).

Thus, in one embodiment, the activity of Rpi-blb2 is further defined asresulting after expression in a plant in necrotic regions that contain alow level of sporulation in experiments as described.

Thus, in one embodiment, the method of the present invention producesplants showing necrotic regions that contain a low level of sporulationor less.

The term “reference” relates to an organism or a part thereof, e.g. acell, which is essentially as identical as possible in genome, proteome,and/or metabolome to the relevant organism or part thereof, e.g. a cell,for example to the plant of the present invention.

Thus, the term “reference” relates for example to an orgamsm or a partthereof, e.g. a cell, which is essentially genetically, proteomically,andlor metabolically identical to the organism of the present inventionor a part thereof but an activity of a specific gene product, e.g.Rpi-blb2, cannot be observed as there is a relevant difference in thereference's genome, proteome or metabolome. Thus, the reference can be aplant or a part thereof which does not express or expresses too littleof a relevant active gene product, e.g. it does not encode a Rpi-blb2 ordoes not transcribe a Rpi-blb2 encoding gene or does not translate anactive Rpi-blb2 mRNA. Thus, the reference does not provide themodification creating an active gene product in a sufficient quantity toresult in an phenotype as described. Whether two plants are essentiallygenetically identical can be tested with assays known to a personskilled in the art, e.g. via fingerprint analysis, e.g. as described inRoldan-Ruiz, Theor. Appl. Genet., 2001, 1138-1150. The expressionpattern of proteins can be tested as described in the art e.g. via gelelectrophoresis (1D, 2D, 3D), mass spectrometric analysis and othermethods. The metabolome can be analysed by the skilled as described inthe art, e.g. via HLPLC, GC, OPLC, LC-MS, GC-MS, LC-MS-MS, and othermethods as described e.g. in Fiehn et al., Nature Biotech, 18 (2000),1157, Raamsdonk et al., Nature Biotech, 19 (2991), 45-50, Buchholz,Anal. Biochem, 295 (2001) 129-137, Soga et al., Anal Chem. 74 (2002)2233-2239.

In order to increase the resistance to a pathogen the reference organismor the part thereof is susceptible to the infection with the pathogen,e.g. a plant pathogen, e.g. P. infestans.

Preferably, the reference is a clone of that organism in which forexample a relevant polynucleotide, e.g. the polynucleotide of theinvention, or an activator, e.g. an activator of a relevant gene productmediating the activity, e.g. an activator increasing the expression of arelevant polynucleotide or a derivate of said polynucleotide, or anactivator of a relevant polypeptide, e.g. of the polypeptide of thepresent invention, and/or a corresponding the relevant gene productencoding vector has been introduced. For example, a preferred referencein the method of the present invention is an organism or a part thereof,which is a clone of the organism or part thereof, e.g. a cell which hasbeen transfected or transformed with the polynucleotide or vector of theinvention.

If the clone as described can not be identified it is state of the artto cleave out, to knock out or to switch off those elements whichessentially mediate the relevant activity, e.g. mediating an increasedRpi-blb2 activity, e.g. mediating an increased expression, in theorganism, e.g. in the plant. It is well known to skilled person, how toreduce or inhibit the activity of a relevant gene product, e.g. byreducing or inhibiting the expression of e.g. Rpi-blb2. Such a clone canthan be compared with an organism produced according to the method ofthe present invention, e.g. a P. infestans resistant, Rpi-blb2expressing genotype.

The term “plant” as used herein refers to all genera and species ofhigher and lower plants of the Plant Kingdom. The term includes themature plants, seed, shoots and seedlings and their derived parts,propagation material, plant organs, tissue, protoplasts, callus andother cultures, for example cell cultures, and any other type of plantcell grouping to give functional or structural units. “Mature plant”refers to a plant at any desired developmental stage beyond that of theseedling. Seedling refers to a young immature plant at an earlydevelopmental stage. “Plant” encompasses all annual and perennialmonocotyledonous and dicotyledonous plants. Preferred within the scopeof the invention are those plants which are employed as foodstuffs orfeeding stuffs, for example monocotyledonous or dicotyledonous genera,in particular species, like the above-described ones, e.g. cerealspecies or members of the Solanaceae family, respectively, mostpreferably potato and tomato.

As known to a person skilled in the art, the method of the presentinvention comprises further selecting those plants in which, as opposedor as compared to the reference plant, the resistance to at least onesaid pathogen exists or is increased.

“Selection” with regard to plants in which—as opposed or as compared tothe reference plant—resistance to at least one pathogen exists or isincreased means all those methods which are suitable for recognizing anexisting or increased resistance to pathogens. These may be symptoms ofpathogen infection but may also comprise the herein described symptomswhich relate to the quality of the plant, the quantity of the yield, thesuitability for use as feeding stuff or foodstuff and the like.

Accordingly, in one embodiment of the method of present invention theRpi-blb2 protein is encoded by a polynucleotide comprising a nucleicacid molecule selected from the group consisting of:

-   a) nucleic acid molecules encoding at least the mature form of the    polypeptide depicted in SEQ ID NO: 2 or 4;-   b) nucleic acid molecules comprising the coding sequence as depicted    in SEQ ID NO: 1 or 3, or 5 or 6 encoding at least the mature form of    the polypeptide;-   c) nucleic acid molecules the nucleotide sequence of which is    degenerate as a result of the genetic code to a nucleotide sequence    of (a) or (b);-   d) nucleic acid molecules encoding a polypeptide derived from the    polypeptide encoded by a polynucleotide of (a) to (c) by way of    substitution, deletion and/or addition of one or several amino acids    of the amino acid sequence of the polypeptide encoded by a    polynucleotide of (a) to (c);-   e) nucleic acid molecules encoding a polypeptide the sequence of    which has an identity of 70% or more to the amino acid sequence of    the polypeptide encoded by a nucleic acid molecule of (a) or (b);-   f) nucleic acid molecules comprising a fragment or a epitope-bearing    portion of a polypeptide encoded by a nucleic acid molecule of any    one of (a) to (e);-   g) nucleic acid molecules comprising a polynucleotide having a    sequence of a nucleic acid molecule amplified from a nucleic acid    library using the primers as listed in Tab 3b, in particular ARF1F    and ARF1R;-   h) nucleic acid molecules encoding a fragment beginning with amino    acid: 1, 30, 50, 100, 200, 300, 500, or 1000 and stopping with amino    acid 1276, 1000, 500, 300, 200, 50, 30, or 1 of a polypeptide    encoded by any one of (a) to (g);-   i) nucleic acid molecules comprising at least 20 nucleotides of a    polynucleotide of any one of (a) or (d);-   j) nucleic acid molecules encoding a polypeptide being recognized by    a monoclonal antibody that have been raised against a polypeptide    encoded by a nucleic acid molecule of any one of (a) to (h);-   k) nucleic acid molecules obtainable by screening an appropriate    library under stringent conditions with a probe having the sequence    of the nucleic acid molecule of any one of (a) to (j) or of a    fragment thereof of at least 15, preferable 30, 60, 100 or more    nucleotides; and-   l) nucleic acid molecules the complementary strand of which    hybridises under stringent conditions with a nucleic acid molecule    of any one of (a) or (k);    or the complementary strand of any one of (a) to (l);    or expressing a polypeptide encoded by a segment or linkage group 6    of Solanum bulbocastanum which co-segregates with a marker selected    from table 3A and which mediates resistance to pathogens, in    particular to pathogens selected from the group consisting of phylum    Oomycetes;

In one embodiment, the polynucleotide of the method of the inventiondoes not consist of the sequence depicted in Seq. ID NO.: 7 and/or 9and/or does not consist of the sequence of a nucleic acid moleculeencoding a protein depicted in Seq. ID NO.: 8 and/or 10.

In one embodiment, the polynucleotide of the method of the inventiondoes not consist of the sequence of a nucleic acid molecule of Mi1.1 orMi1.2 and/or of a nucleic acid molecule encoding a Mi1.1 or Mi1.2protein.

Thus, in one embodiment, the polynucleotide of method of the presentinvention may not consist of the sequences shown in Rossi et al. 1998,PNAS USA 95:9750-9754, Milligan et al., 1998. Plant Cell 10:1307-1319;and/or WO 9806750. A comparison of the sequences of Rpi-blb2, Mi1.1 andMi1.2 is shown in FIGS. 15 to 17.

The term “linkage group” as used herein relates to two or more traitsand/or loci and/or genes and/or markers that tend to be inheritedtogether as a consequence of an association between said traits and/orloci and/or genes and/or markers. The closer together the traits and/orloci and/or genes and/or markers are, the lower the probability thatthey will be separated during DNA repair or replication processes suchas mitosis or meiosis in eukaryotes, and hence the greater theprobability that they will be inherited together. There are as manylinkage groups as there are homologous pairs of chromosomes.

The term “linkage group 6” relates to a linkage group of potato ortomato which is affiliated to chromosome 6, such affiliation establishedby identifying markers of known chromosomal position based on workpublished by Bernatzky and Tanksley (1986) and Tanksley et al. (1992).Linkage groups bear the same numbers as their respective chromosomes. Intomato, the chromosomes are numbered according to their length measuredin pachytene. Such numbers have been applied by Barton (1950);chromosome 1 is the longest, chromosome 12, the shortest. In addition tolength, such features as positions of centromere and amount anddistribution of heterochromatin serve to identify each chromosome. Shortarms are symbolized by “S”, long ones by “L”; thus “1S” designates theshort arm of chromosome 1; as e.g. in Barton, D. W. (1950) AmericanJournal of Botany. 37,639-643, Bernatzky, R. and Tanksley, S. D. (1986)Genetics 112, 887-898, Tanksley, S. D., et al., (1992) Genetics 132,1141-1160.

The term “co-segregation” as used herein relates to the tendency for twoor more closely linked traits and/or loci and/or genes and/or markers tobe inherited together.

For example, the more concrete region of chromosome 6 that co-segregateswith Rpi-blb2 is the short arm that, in tomato, bears the morphologicalmarker Mi.

Accordingly, in one embodiment the present invention relates to themethod of the present invention, wherein the Rpi-blb2 protein is encodedby the polynucleotide of the present invention, e.g. encoded by apolynucleotide shown in Seq. ID. 1 or 3 or 5 or 6 or a fragment thereof.

On basis of a BLASTX search the genes with the highest homologyidentified to the identified Rpi-blb2 sequences were the Mi1.1- andMi1.2-genes and proteins; see FIGS. 15 to 17. Both genes have a highidentity to the sequence depicted in Seq. ID NO.: 1 or 3 or 5 or 6 butdo not confer resistance to the plant pathogen of the phylum Oomycetes.Therefore the activity of Mi1.1 and Mi1.2 is an other activity as theactivity of the polypeptid of the present invention. The sequence ofMi1.1 and Mi1.2 ORF and encoded proteins is herein shown in Seq. ID NO.:7 to 10. Further, the application EP 401764.4 relates to the Mi-genes.The sequence of prior art Mi1.1- and Mi1.2-genes is excluded from thepolynucleotide of the present invention, in particular Seq. ID NO.: 7and 9 are excluded. Also included may be polynucleotide sequencesencoding the polypeptide of Seq. ID NO.: 8 or 10, Thus, in an embodimentalso sequences encoding the Mi1.1 and Mi1.2 protein are excluded.Proteins with a lower homology to the polypeptide encoded by thepolynucleotide of the present invention are Hero Resistance proteins 1and 2 (Genbank AccNo.: gi26190252 and gi26190254), Tospovirus resistanceproteins A, B, C, D and E [Genbank AccNos.: gi15418709, gi15418710,gi15418712, gi15418713, gi15418714]; R1 [GenbankAccNo.: gi17432423] andPrf [Genbank AccNo.: gi8547237] which sequences or encoded sequences areas well excluded from the sequences of the present invention.

The terms “gene(s)”, “polynucleotide”, “nucleic acid sequence”,“nucleotide sequence”, or “nucleic acid molecule(s)” as used hereinrefer to a polymeric form of nucleotides of any length, eitherribonucleotides or deoxyribonucleotides. This term refers only to theprimary structure of the molecule.

Thus, this term includes double- and single-stranded DNA, and RNA. Italso includes known types of modifications, for example, methylation,“caps” substitution of one or more of the naturally occurringnucleotides with an analogue. Preferably, the DNA sequence of theinvention comprises a coding sequence encoding the herein definedpolypeptide.

A “coding sequence” is a nucleotide sequence which is transcribed intomRNA and/or translated into a polypeptide when placed under the controlof appropriate regulatory sequences. The boundaries of the codingsequence are determined by a translation start codon at the 5′-terminusand a translation stop codon at the 3′-terminus. A coding sequence caninclude, but is not limited to mRNA, cDNA, recombinant nucleotidesequences or genomic DNA, while introns may be present as well undercertain circumstances.

By “hybridising” it is meant that such nucleic acid molecules hybridiseunder conventional hybridisation conditions, preferably under stringentconditions such as described by, e.g., Sambrook (Molecular Cloning; ALaboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1989)). An example of one such stringenthybridisation condition is hybridisation at 4×SSC at 65° C., followed bya washing in 0.1×SSC at 65° C. for one hour. Alternatively, an exemplarystringent hybridisation condition is in 50% formamide, 4×SSC at 42° C.Further, the conditions during the wash step can be selected from therange of conditions delimited by low-stringency conditions(approximately 2×SSC at 50° C.) and high-stringency conditions(approximately 0.2×SSC at 50° C., preferably at 65° C.) (20×SSC: 0.3Msodium citrate, 3M NaCl, pH 7.0). In addition, the temperature duringthe wash step can be raised from low-stringency conditions at roomtemperature, approximately 22° C., to higher-stringency conditions atapproximately 65° C. Both of the parameters salt concentration andtemperature can be varied simultaneously, or else one of the twoparameters can be kept constant while only the other is varied.Denaturants, for example formamide or SDS, may also be employed duringthe hybridisation. In the presence of 50% form amide, hybridisation ispreferably effected at 42° C. Some further examples of conditions forhybridisation and wash step are shown herein below:

-   (1) Hybridisation conditions can be selected, for example, from the    following conditions:-   a) 4×SSC at 65° C.,-   b) 6×SSC at 45° C.,-   c) 6×SSC, 100 mg/ml denatured fragmented fish sperm DNA at 68° C.,-   d) 6×SSC, 0.5% SDS, 100 mg/ml denatured salmon sperm DNA at 68° C.,-   e) 6×SSC, 0.5% SDS, 100 mg/ml denatured fragmented salmon sperm DNA,    50% formamide at 42° C.,-   f) 50% formamide, 4×SSC at 42° C.,-   g) 50% (vol/vol) formamide, 0.1% bovine serum albumin, 0.1% Ficoll,    0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer pH 6.5, 750    mM NaCl, 75 mM sodium citrate at 42° C.,-   h) 2× or 4×SSC at 50° C. (low-stringency condition), or-   i) 30 to 40% formamide, 2× or 4×SSC at 42° C. (low-stringency    condition).-   (2) Wash steps can be selected, for example, from the following    conditions:-   a) 0.015 M NaCl/0.0015 M sodium citrate/0.1% SDS at 50° C.-   b) 0.1×SSC at 65° C.-   c) 0.1×SSC, 0.5% SDS at 68° C.-   d) 0.1×SSC, 0.5% SDS, 50% formamide at 42° C.-   e) 0.2×SSC, 0.1% SDS at 42° C.-   f) 2×SSC at 65° C. (low-stringency condition).

In one embodiment of the present invention, the polynucleotide of theinvention comprises a polynucleotide which hybridises to a nucleic acidmolecule comprising or consisting of a nucleic acid molecule having thesequence shown in Seq ID No. 1 or 3 or 5 or 6 or a fragment thereof. Thefragment comprises or consists preferably of 15, 20, 30, 40, 70, 100,300, 500, 700, 1000 or more residues of Seq ID No. 1 or 3 or 5 or 6.

In a preferred embodiment, the polynucleotide of the invention comprisesa polynucleotide which hybridises under “stringent” hybridisationconditions with a nucleic acid molecule comprising or consisting of anucleic acid molecule having the sequence shown in Seq ID No. 1 or 3 or5 or 6 or a fragment thereof.

The term “under stringent hybridisation conditions” as used hereinrefers to any of the herein mentioned stringent hybridisationconditions. In a further embodiment, the term “under stringenthybridisation conditions” refers to the hybridisation conditionsmentioned in the examples or used in Sambrook (Molecular Cloning; ALaboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1989).

In one preferred embodiment, the term “under stringent hybridisationconditions” as used herein refers to all of the herein mentionedstringent hybridisation conditions, meaning that a polynucleotidehybridises under all mentioned stringent conditions.

Rpi-blb2 derived from other organisms, may be encoded by other DNAsequences which hybridise to the sequences shown in Seq ID No. 1 or 3 or5 or 6 under relaxed hybridisation conditions and which code onexpression for peptides having the activity of Rpi-blb2. Further, someapplications have to be performed at low stringency hybridisationconditions, without any consequences for the specificity of thehybridisation. For example, a Southern blot analysis of total DNA couldbe probed with a polynucleotide of the present invention and washed atlow stringency (55° C. in 2×SSPE, 0.1% SDS). The hybridisation analysiscould reveal a simple pattern of only genes encoding Rpi-blb2. A furtherexample of such low-stringent hybridisation conditions are 4×SSC at 50°C. or hybridisation with 30 to 40% formamide at 42° C. Such moleculescomprise those which are fragments, analogues or derivatives of Rpi-blb2of the invention and differ, for example, by way of amino acid and/ornucleotide deletion(s), insertion(s), substitution (s), addition(s)and/or recombination (s) or any other modification(s) known in the arteither alone or in combination from the above-described amino acidsequences or their underlying nucleotide sequence(s). However, it ispreferred to use high stringency hybridisation conditions.

The term “homology” means that the respective nucleic acid molecules orencoded proteins are functionally and/or structurally equivalent. Thenucleic acid molecules that are homologous to the nucleic acid moleculesdescribed above and that are derivatives of said nucleic acid moleculesare, for example, variations of said nucleic acid molecules whichrepresent modifications having the same biological function, inparticular encoding proteins with the same or substantially the samebiological function. They may be naturally occurring variations, such assequences from other plant varieties or species, or mutations. Thesemutations may occur naturally or may be obtained by mutagenesistechniques. The allelic variations may be naturally occurring allelicvariants as well as synthetically produced or genetically engineeredvariants. Structurally equivalents can, for example, identified bytesting the binding of said polypeptide to antibodies. Structurallyequivalent have the similar immunological characteristic, e.g. comprisesimilar epitopes.

The terms “fragment”, “fragment of a sequence” or “part of a sequence”mean a truncated sequence of the original sequence referred to. Thetruncated sequence (nucleic acid or protein sequence) can vary widely inlength; the minimum size being a sequence of sufficient size to providea sequence with at least a comparable function and/or activity of theoriginal sequence referred to, while the maximum size is not critical.In some applications, the maximum size usually is not substantiallygreater than that required to provide the desired activity and/orfunction(s) of the original sequence.

Typically, the truncated amino acid sequence will range from about 5 toabout 1260 amino acids in length. More typically, however, the sequencewill be a maximum of about 1000 amino acids in length, preferably amaximum of about 500 or 100 amino acids. It is usually desirable toselect sequences of at least about 10, 12 or 15 amino acids, up to amaximum of about 20 or 25 amino acids.

The term “epitope” relates to specific immunoreactive sites within anantigen, also known as antigenic determinates. These epitopes can be alinear array of monomers in a polymeric composition—such as amino acidsin a protein—or consist of or comprise a more complex secondary ortertiary structure. Those of skill will recognize that all immunogens(i.e., substances capable of eliciting an immune response) are antigens;however, some antigen, such as haptens, are not immunogens but may bemade immunogenic by coupling to a carrier molecule. The term “antigen”includes references to a substance to which an antibody can be generatedand/or to which the antibody is specifically immunoreactive. In oneembodiment the present invention relates to a epitope of Rpi-blb2.

The term “one or several amino acids” relates to at least one amino acidbut not more than that number of amino acids which would result in ahomology of below 70% identity. Preferably, the identity is more than75% or 80%, more preferred are 85%, 90% or 95%, even more preferred are96%, 97%, 98%, or 99% identity.

The terms “polynucleotide” and “nucleic acid molecule” also relate to“isolated” polynucleotides or nucleic acids molecules. An “isolated”nucleic acid molecule is one which is separated from other nucleic acidmolecules which are present in the natural source of the nucleic acid.Preferably, an “isolated” nucleic acid is free of sequences whichnaturally flank the nucleic acid (i.e., sequences located at the 5′ and3′ ends of the nucleic acid) in the genomic DNA of the organism fromwhich the nucleic acid is derived. For example, in various embodiments,the polynucleotide of the present invention can contain less than about5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb or less of nucleotidesequences which naturally flank the nucleic acid molecule in genomic DNAof the cell from which the nucleic acid is derived. Moreover, thepolynucleotides of the present invention, in particular an “isolated”nucleic acid molecule, such as a cDNA molecule, can be substantiallyfree of other cellular material, or culture medium when produced byrecombinant techniques, or chemical precursors or other chemicals whenchemically synthesized.

Further, the polynucleotide of the invention comprises a nucleic acidmolecule which is a complement of one of the nucleotide sequences ofabove mentioned polynucleotides or a portion thereof. A nucleic acidmolecule which is complementary to one of the nucleotide sequences shownin SEQ ID No:1 or 3 or 5 or 6 is one which is sufficiently complementaryto one of the nucleotide sequences shown in SEQ ID No: 1 or 3 or 5 or 6such that it can hybridise to one of the nucleotide sequences shown inSEQ ID No: 1 or 3 or 5 or 6, thereby forming a stable duplex.

The polynucleotide of the invention comprises a nucleotide sequencewhich is at least about 70%, preferably at least about 75%, morepreferably at least about 80%, 90%, or 95%, and even more preferably atleast about 96%, 97%, 98%, 99% or more homologous to a nucleotidesequence shown in SEQ ID No: 1 or 3 or 5 or 6, or a portion thereof. Thepolynucleotide of the invention comprises a nucleotide sequence whichhybridises, preferably hybridises under stringent conditions as definedherein, to one of the nucleotide sequences shown in SEQ ID No: 1 or 3 or5 or 6, or a portion thereof.

Moreover, the polynucleotide of the invention can comprise only aportion of the coding region of one of the sequences in SEQ ID No: 1 or3 or 5 or 6, for example a fragment which can be used as a probe orprimer or a fragment encoding a biologically active portion of theRpi-blb2 protein coding gene. The nucleotide sequences determined fromthe cloning of the present Rpi-blb2 protein encoding gene allows for thegeneration of probes and primers designed for use in identifying and/orcloning its homologues in other cell types and organisms. Theprobe/primer typically comprises substantially purifiedoligonucleotides. The oligonucleotide typically comprises a region ofnucleotide sequence that hybridises under stringent conditions to atleast about 12, 15 preferably about 20 or 25, more preferably about 40,50 or 75 consecutive nucleotides of a sense strand of one of thesequences set forth, e.g., in SEQ ID No. No: 1 or 3 or 5 or 6, ananti-sense sequence of one of the sequences, e.g., set forth in SEQ IDNo.: 1 or 3 or 5 or 6, or naturally occurring mutants thereof. Primersbased on a nucleotide of invention can be used in PCR reactions to cloneRpi-blb2 homologues, e.g. as the primers described in the examples ofthe present invention, e.g. as shown in tab 3a or 3b, preferably theprimers ARF1F and ARF1R are used. A PCR with the primers univ24R anduniv14L will result in a fragment of Rpi-blb2 which can be used asdescribed herein. Said primer sets are interchangeable. The personskilled in the art knows to combine said primers to result in thedesired product, e.g. in a full length clone or a partial sequence.Probes based on the Rpi-blb2 nucleotide sequences can be used to detecttranscripts or genomic sequences encoding the same or homologousproteins. The probe can further comprise a label group attached thereto,e.g. the label group can be a radioisotope, a fluorescent compound, anenzyme, or an enzyme cofactor. Such probes can be used as a part of agenomic marker test kit for identifying cells which express a Rpi-blb2,such as by measuring a level of a Rpi-blb2-encoding nucleic acidmolecule in a sample of cells, e.g., detecting Rpi-blb2 mRNA levels ordetermining whether a genomic Rpi-blb2 gene has been mutated or deleted.

The polynucleotide of the invention encodes a polypeptide or portionthereof which includes an amino acid sequence which is sufficientlyhomologous to the amino acid sequence of SEQ ID No: 2 or 4 such that theprotein or portion thereof maintains the ability to participate inresistance to pathogens, in particular a Rpi-blb2 protein activity asdescribed in the examples in plants. As used herein, the language“sufficiently homologous” refers to proteins or portions thereof whichhave amino acid sequences which include a minimum number of identical orequivalent (e.g., an amino acid residue which has a similar side chainas an amino acid residue in one of the sequences of the polypeptide ofthe present invention), amino acid residues to an amino acid sequence ofSeq. ID No.: 2 or 4 such that the protein or portion thereof is able toparticipate in the resistance of plants to said pathogens. Examples of aRpi-blb2 protein activity are described herein. Thus, the function of aRpi-blb2 protein contributes either directly or indirectly to theresistance to plant pathogens, preferably to the pathogens mentionedherein, more preferred to P. infestans.

The protein is at least about 70%, preferably at least about 75%, andmore preferably at least about 80%, 90%, 95%, and most preferably atleast about 96%, 97%, 98%, 99% or more homologous to an entire aminoacid sequence of SEQ ID No: 2 or 4.

Portions of proteins encoded by the polynucleotide of the invention arepreferably biologically active.

As mentioned herein, the term “biologically active portion” is intendedto include a portion, e.g., a domain/motif, that confers resistance toan oomycete plant pathogen and/or Bemisia tabaci and/or aphids or has animmunological activity such that it binds to an antibody bindingspecifically to Rpi-blb2 protein or it has an activity as set forth inthe Examples or as described herein.

Additional nucleic acid fragments encoding biologically active portionsof the polypeptide of the present invention can be prepared by isolatinga portion of one of the sequences in SEQ ID No: 1 or 3 or 5 or 6,expressing the encoded portion of the Rpi-blb2 protein or peptide (e.g.,by recombinant expression in vitro) and assessing the activity of theencoded portion of the protein.

The invention further encompasses polynucleotides that differ from oneof the nucleotide sequences shown in SEQ ID No: 1 or 3 or 5 or 6 (andportions thereof due to degeneracy of the genetic code and thus encode aRpi-blb2 polypeptide as that encoded by the sequences shown in SEQ IDNo: 2 or 4. Further the polynucleotide of the invention has a nucleotidesequence encoding a protein having an amino acid sequence shown in SEQID No: 2 or 4. In a still further embodiment, the polynucleotide of theinvention encodes a full length protein which is substantiallyhomologous to an amino acid sequence of SEQ ID No: 2 or 4.

In addition, it will be appreciated by those skilled in the art that DNAsequence polymorphisms that lead to changes in the amino acid sequencesmay exist within a population (e.g., the S. bulbocastanum population).Such genetic polymorphism in the Rpi-blb2 gene may exist amongindividuals within a population due to natural variation.

As used herein, the terms “gene” and “recombinant gene” refer to nucleicacid molecules comprising an open reading frame encoding a Rpi-blb2,preferably a S. bulbocastanum Rpi-blb2. Such natural variations cantypically result in 1-5% variance in the nucleotide sequence of theRpi-blb2 gene. Any and all such nucleotide variations and resultingamino acid polymorphisms in Rpi-blb2 that are the result of naturalvariation and that do not alter the functional activity of Rpi-blb2 areintended to be within the scope of the invention.

Polynucleotides corresponding to natural variants and non-S.bulbocastanum homologues of the Rpi-blb2 cDNA of the invention can beisolated based on their homology to S. bulbocastanum Rpi-blb2polynucleotides disclosed herein using the polynucleotide of theinvention, or a portion thereof, as a hybridisation probe according tostandard hybridisation techniques under stringent hybridisationconditions. Accordingly, in another embodiment, a polynucleotide of theinvention is at least 20 nucleotides in length. Preferably it hybridisesunder stringent conditions to the nucleic acid molecule comprising anucleotide sequence of the polynucleotide of the present invention, e.g.SEQ ID No: 1 or 3 or 5 or 6. In other embodiments, the nucleic acid isat least 20, 30, 50, 100, 250 or more nucleotides in length. The term“hybridises under stringent conditions” is defined above and is intendedto describe conditions for hybridisation and washing under whichnucleotide sequences at least 65% identical to each other typicallyremain hybridised to each other. Preferably, the conditions are suchthat sequences at least about 70%, more preferably at least about 75% or80%, and even more preferably at least about 85%, 90% or 95% or moreidentical to each other typically remain hybridised to each other.Preferably, polynucleotide of the invention that hybridises understringent conditions to a sequence of SEQ ID No: 1 or 3 or 5 or 6corresponds to a naturally-occurring nucleic acid molecule.

As used herein, a “naturally-occurring” nucleic acid molecule refers toan RNA or DNA molecule having a nucleotide sequence that occurs innature (e.g., encodes a natural protein). Preferably, the polynucleotideencodes a natural S. bulbocastanum Rpi-blb2.

In addition to naturally-occurring variants of the Rpi-blb2 sequencethat may exist in the population, the skilled artisan will furtherappreciate that changes can be introduced by mutation into a nucleotidesequence of the polynucleotide encoding Rpi-blb2, thereby leading tochanges in the amino acid sequence of the encoded Rpi-blb2, withoutaltering the functional ability of the Rpi-blb2. For example, nucleotidesubstitutions leading to amino acid substitutions at “non-essential”amino acid residues can be made in a sequence of the polynucleotideencoding Rpi-blb2, e.g. SEQ ID No: 1 or 3 or 5 or 6. A “non-essential”amino acid residue is a residue that can be altered from the wild-typesequence of the Rpi-blb2 protein without altering the activity of saidRpi-blb2 protein, hereas an “essential” amino acid residue is requiredfor Rpi-blb2 protein activity. Other amino acid residues, however,(e.g., those that are not conserved or only semi-conserved in the domainhaving Rpi-blb2 activity) may not be essential for activity and thus arelikely to be amenable to alteration without altering Rpi-blb2 activity.

Accordingly, a person skilled in the art knows that the codon usagebetween organisms can differ. Therefore he will adapt the codon usage inthe polynucleotide of the present invention to the usage of the organismin which the polynucleotide or polypeptide is expressed.

Accordingly, the invention relates to polynucleotides encoding Rpi-blb2that contain changes in amino acid residues that are not essential forRpi-blb2 activity. Such Rpi-blb2s differ in amino acid sequence from asequence contained in SEQ ID No: 2 or 4 yet retain the Rpi-blb2 activitydescribed herein. The polynucleotide can comprise a nucleotide sequenceencoding a polypeptide, wherein the polypeptide comprises an amino acidsequence at least about 70% identical to an amino acid sequence of SEQID No: 2 or 4 and is capable of participation in the resistance to aplant pathogen. Preferably, the protein encoded by the nucleic acidmolecule is at least about 70% identical to the sequence in SEQ ID No: 2or 4, more preferably at least about 75% identical to one of thesequences in SEQ ID No: 2 or 4, even more preferably at least about 80%,90%, 95% homologous to the sequence in SEQ ID No: 2 or 4, and mostpreferably at least about 96%, 97%, 98%, or 99% identical to thesequence in SEQ ID No: 2 or 4.

To determine the percent homology of two amino acid sequences (e.g., oneof the sequences of Seq. ID No.: 2 or 4 and a mutant form thereof) or oftwo nucleic acids, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in the sequence of one protein ornucleic acid for optimal alignment with the other protein or nucleicacid). The amino acid residues or nucleotides at corresponding aminoacid positions or nucleotide positions are then compared. When aposition in one sequence (e.g., one of the sequences of SEQ ID No: 2 or4) is occupied by the same amino acid residue or nucleotide as thecorresponding position in the other sequence (e.g., a mutant form of thesequence selected), then the molecules are homologous at that position(i.e., as used herein amino acid or nucleic acid “homology” isequivalent to amino acid or nucleic acid “identity”). The percenthomology between the two sequences is a function of the number ofidentical positions shared by the sequences (i.e., % homology=numbers ofidentical positions/total numbers of positions×100).

Homology can be calculated by comparison with the aid of the programalgorithm GAP (Wisconsin Package Version 10.0, University of Wisconsin,Genetics Computer Group (GCG), Madison, USA; Altschul et al. (1997)Nucleic Acids Res. 25:3389 et seq.), setting the following parameters:

Gap weight: 50 Length weight: 3 Average match: 10 Average mismatch: 0

For example a sequence which has at least 80% homology with sequence SEQID NO: 1 at the nucleic acid level is understood as meaning a sequencewhich, upon comparison with the sequence SEQ ID NO: 1 by the aboveprogram algorithm with the above parameter set, has at least 80%homology.

Homology between two polypeptides is understood as meaning the identityof the amino acid sequence over in each case the entire sequence lengthwhich is calculated by comparison with the aid of the program algorithmGAP (Wisconsin Package Version 10.0, University of Wisconsin, GeneticsComputer Group (GCG), Madison, USA), setting the following parameters:

Gap weight: 8 Length weight: 2 Average match: 2,912 Average mismatch:−2,003

For example a sequence which has at least 80% homology with sequence SEQID NO: 2 at the protein level is understood as meaning a sequence which,upon comparison with the sequence SEQ ID NO: 2 by the above programalgorithm with the above parameter set, has at least 80% homology.

In the present application, the homology was determined with the programclustalW, choose sequence analyses and choose option clustalW (multiplesequence alignments). All options were performed under standardconditions, as follows:

alignment: full; output format: aln w/numbers; output order: aligned;color alignment: no; ktup (word size): def; window length: def; scoretype: percent; topdiag: def; pairgap: def; matrix: def; gap open: def;end gaps: def; gap extension: def; gap distances: def; cpu mode: single;tree graph/type: cladogram; tree graph/distances: hide; phylogenetictree/tree type: none; phylogenetic tree/correct dist.: off; phylogenetictree/ignore gaps: off. Therefore a Homology calculation according toclustalW is preferred.

Functional equivalents derived from one of the polypeptides as shown inSEQ ID NO: 2 or 4 according to the invention by substitution, insertionor deletion have at least 70%, preferably at least 80%, by preference atleast 90%, especially preferably at least 95%, very especiallypreferably at least 98%, homology with one of the polypeptides as shownin SEQ ID NO: 2 or 4 according to the invention and are distinguished byessentially the same properties as the polypeptide as shown in SEQ IDNO: 2 or 4.

Functional equivalents derived from the nucleic acid sequence as shownin SEQ ID NO: 1 or 3 or 5 or 6 according to the invention bysubstitution, insertion or deletion have at least 70%, preferably atleast 80%, by preference at least 90%, especially preferably at least95%, very especially preferably at least 98%, homology with one of thepolypeptides as shown in SEQ ID NO: 2 or 4 according to the inventionand encode polypeptides having essentially the same properties as thepolypeptide as shown in SEQ ID NO: 2 or 4.

“Essentially the same properties” of a functional equivalent is aboveall understood as meaning conferring a pathogen-resistant phenotype orconferring or increasing the resistance to at least one pathogen whileincreasing the amount of protein, activity or function of saidfunctional Rpi-blb2 equivalent in a plant or in a tissue, part or cellsof the same. The sporulation and lesion phenotype after infection incombination with said increase of the amount of protein, activity orfunction of the functional equivalent is furthermore understood as anessential property.

A nucleic acid molecule encoding a Rpi-blb2 homologous to a proteinsequence of SEQ ID No: 2 or 4 can be created by introducing one or morenucleotide substitutions, additions or deletions into a nucleotidesequence of the polynucleotide of the present invention, in particularof SEQ ID No: 1 or 3 or 5 or 6 such that one or more amino acidsubstitutions, additions or deletions are introduced into the encodedprotein. Mutations can be introduced into the sequences of, e.g., SEQ IDNo: 1 or 3 or 5 or 6 by standard techniques, such as site-directedmutagenesis and PCR-mediated mutagenesis. Preferably, conservative aminoacid substitutions are made at one or more predicted non-essential aminoacid residues. A “conservative amino acid substitution” is one in whichthe amino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Thus, a predicted nonessential amino acid residue in aRpi-blb2 is preferably replaced with another amino acid residue from thesame family. Alternatively, in another embodiment, mutations can beintroduced randomly along all or part of a Rpi-blb2 coding sequence,such as by saturation mutagenesis, and the resultant mutants can bescreened for a Rpi-blb2 activity described herein to identify mutantsthat retain Rpi-blb2 activity. Following mutagenesis of one of thesequences of SEQ ID No: 1 or 3 or 5 or 6, the encoded protein can beexpressed recombinantly and the activity of the protein can bedetermined using, for example, assays described herein (see Examples).

In one embodiment, in the method of present invention the activity ofRpi-blb2 protein and of a further resistance protein is increased.

It is expected, that under field conditions the presence of more thanone resistance gene is beneficial, in particular genes conferringresistance to the same pathogen. In case a pathogen isolate, e.g. a P.infestans race, is present that is able to overcome resistance of one ofthe R-genes, the other one or more R-gene(s) is/are still functionalmaking it impossible to infect the plant. The present of two undefeated

R-genes strongly reduces the chance that a pathogen, in particular a P.infestans race, is able to mutate into a race that can overcome two ormore R-genes.

In the following “resistance polypeptide” or “resistance protein”relates to a polypeptide which (increased) activity will conferresistance to a susceptible genotype (“wild type” or “reference”).Accordingly, Rpi-blb2 is a resistance protein as well as e.g. Rpi-blb(or RB or Sbu1). A “further resistance protein” relates to an otherresistance protein than the protein of the present invention, whereasthe term “resistance protein” comprises the polypeptid of the presentinvention as well as one or more further resistance protein(s). It isfurther understood, that the term “and a further resistance protein”relates to one or more further resistance proteins. Thus, the activityof one or more resistance proteins can be increased. Further resistanceproteins are described below. However, generally any other knownresistance protein can be co-expressed with the polypeptid of thepresent invention or its activity can be increased by any of the methodsdescribed herein for Rpi-blb2.

In a preferred embodiment, the further resistance protein comprises aLRR domain and a P-loop.

The cloning and molecular characterisation of over 30 plant diseaseresistance (R) genes conferring resistance to bacteria, fungi,oomycetes, viruses, nematodes, or insects has allowed theirclassification in structural classes regardless of pathogen specificity(reviewed in Dangl and Jones, 2001). The most abundant class ofcharacterised R genes, comprising about 0.5 percent of the genespredicted in the Arabidopsis genome, is predicted to encodeintracellular proteins that carry leucine-rich repeat (LRR) andnucleotide-binding site (NBS) domains, motifs also found in otherreceptor and signal transduction proteins. NBS-LRR R proteins differprimarily at the N-terminus that either exhibits sequence similarity tothe Drosophila Toll protein and the mammalian interleukin-1 receptordomain (TIR-NBS-LRR), or code for a coiled-coils structure (CC-NBS-LRR),sometimes in the form of a leucine zipper (LZ-NBS-LRR). Although maybemembrane associated, NBS-LRR proteins are predicted to be cytoplasmic.In contrast, two other classes of R proteins that carry LRRs arepredicted to span the cell membrane, with an extracellular LRR domain:the LRR-transmembrane (LRR-TM) Cf proteins and the LRR-TM-kinase Xa21protein. Characterised R proteins that lack LRRs are the Pto gene fromtomato, the Hs1^(pro-1) gene from beet, the mlo gene from barley, theRpw8 genes from Arabidopsis and the Rpg1 gene from barley.

According to the gene-for-gene hypothesis, disease resistance followsperception by plant R proteins of pathogen effector molecules withavirulence (Avr) function, thereby initiating through some kind ofelicitor recognition complex, signal transduction pathways leading to ahypersensitive response (HR). In common with other receptors it isgenerally considered that NBS-LRR R proteins have a modular structurewith separate recognition and signalling domains, whereby the LRR is thecandidate recognition domain and the N-terminal region including theNBS, the major signalling domain. Functional analysis of recombinant Rproteins indicates that recognition specificity indeed resides in theLRR. Moreover, the LRR is the most variable region in closely relatedNBS-LRR proteins and is under selection to diverge. However, evidence isaccumulating that LRRs also contribute to signalling through negativeregulation involving putative intramolecular interactions. Currently,five R genes have been cloned from potato, including two R genesconferring resistance to late blight, and all belong to theCC/LZ-NBS-LRR class of plant R genes. While the S. demissum derived R1gene confers race specific resistance to late blight, the recentlycloned S. bulbocastanum derived gene Rpi-blb (or RB or Sbu1) confersfull resistance to a range of P. infestans isolates carrying multiplevirulence factors and race-specificity has not yet been demonstrated.Furthermore, as described before, progeny plants of somatic hybridscontaining Rpi-blb were unaffected by late blight on field experimentsin Mexico, where nearly every race of the fungus is found. Throughcomplementation of the susceptible phenotype in cultivated potato andtomato the potential of interspecific transfer of broad-spectrum lateblight resistance to cultivated Solanaceae from sexually incompatiblehost species by transformation with single cloned R genes wasdemonstrated. U.S. Pat. No. 6,127,607 describes resistance proteins withLRR domains and P-loops. The content of U.S. Pat. No. 6,127,607 isherewith incorporated by reference. In particular columns 6 to 8 andcol. 11 describe LRR domains and P-loops. Furthermore Song, 2003, PNAS100 (16), 9128-9133 shows a comparison of Rpi-blb LRR motifs in FIG. 4and gives on pages 9132 an over-view about LRR domains. The domains ofthe polypeptid of the present invention are shown in FIG. 14 as well asin FIG. 15.

Preferably the activity of one or more resistance protein(s) selectedfrom the group consisting of Rpi-blb (synonym RB or Sbu1), Rpi-ABPT1,Rpi-blb3, Rpi-mcd, R1, R-ber (synonym R12), Rpi1, R2, R3a, R3b, R4, R5,R6, R7, R8, R9, R10, R11, Ph-1, Ph-2 and Ph-3 is increased. Preferred isthat in addition to Rpi-blb2 at least also the Rpi-blb activity isincreased.

In one embodiment of the present invention, the expression of an, e.g.transgenic, Rpi-blb2 protein is increased and further a transgenicresistance gene's expression is increased. The resistance proteincoexpressed with the Rpi-blb2 (or RB or Sbu1) is preferably one of theresistance proteins mentioned herein, in particular Rpi-blb, Rpi-ABPT1,Rpi-blb3, R1, Rpi1, R-ber, Rpi-mcd, R2, R3a, R3b, R6, R7, Ph-1, Ph-2 orPh-3 but can also be one of the others resistance to plant pathogensconferring proteins known to a person skilled in the art.

As mentioned, the term “increased expression” according to thisinvention also includes a de novo-Expression of a polynucleotide orpolypeptide.

Most preferred is an increase of resistance via coexpression of thepolypeptid of the present invention together with Rpi-blb. Rpi-blb andRpi-blb2 provide both full resistance in detached leaf assays to P.infestans isolates as described in the examples, and in Song 2003, PNAS100 (16), 9128.

Said resistance conferring genes are for example described in

-   RB or Sbu1 (synonym of Rpi-blb): AY336128 [gi: 32693280], (Song et    al., 2003). BAC clones 177 013 and CB3A14 comprising the Rpi-blb    gene have been deposited in GenBank with accession nos AY303171 and    AY303170.-   R1: AF447489 [gi: 9117432422], (Ballvora et al., 2002)-   Rpi1: Kuhl, J. C., Hanneman, R. E., and Havey, M. J., (2201)    Characterization and mapping of Rpi1, a late blight resistance locus    from diploid (1EBN) Mexican Solanum pinnatisectum. Molecular genet.    Genomics 265: 977-985.-   R-ber: Ewing, E. E., Simko, I., Smart, C. D., Bonierbale, M. W.,    Mizubuti, E. S. G., May, G. D., and Fry, W. E., (2000) Genetic    mapping from field tests of qualitative and quantitative resistance    to Phytophthora infestans in a population derived from Solanum    tuberosum and Solanum berthaultii. Molecular breeding 6:25-36.-   R2: Li, X., vanEck, H. J., vanderVoort, J. N. A. M., Huigen, D. J.,    Stam, P., and Jacobsen, E. (1998) Autotetraploids and genetic    mapping using common AFLP markers: the R2 allele conferring    resistance to Phytophthora infestans mapped on potato chromosome 4.    Theoretical and Applied Genetics 96 (8): 1121-112.-   R3, R6, R7: Elkharbotly, A., Palominosanchez, C., Salamini, F.,    Jacobsen, E., and Gebhardt, C. (1996) R6 and R7 alleles of potato    conferring race-specific resistance to Phytophthora infestans (Mont)    de Bary identified genetic loci clustering with the R3 locus on    chromosome XI. Theoretical and Applied. Genetics 92 (7): 880-884.-   Ph-1: Bonde and Murphy (1952) Main Agric. Exp. Stn. Bull. No 497-   Ph-2: Moreau, P., Thoquet, P., Olivier, J., Laterrot, H., and    Grimsley, N. H. (1998) Genetic mapping of Ph-2, a single locus    controlling partial resistance to Phytophthora infestans in tomato.    Molecular Plant Microbe Interactions 11 (4): 259-269.-   Ph-3: Chunwongse, J., Chunwongse, C., Black, L., and    Hanson, P. (2002) Molecular mapping of the Ph-3 gene for late blight    resistance in tomato. Journal of Horticultural Science &    Biotechnology 77 (3): 281-286.-   Rpi-blb3, Rpi-ABPT1 and Rpi-mcd: Park, T. H., Van der Vossen, E.,    Vleeshouwers, V. G. A. A., Tan, A., Visser, R. G. F. and Van    Eck, H. J. 2004. Major resistance genes for tuber and leaf    resistance to Phytophthora infestans in potato: An outline of a PhD    project. Crop Functional Genomics 2004, July 2004, Jeju, Korea, page    93.-   R3a and R3b: Huang, S., Vleeshouwers, V. G. A. A., Werij, J. S.,    Hutten, R. C. B., Van Eck, H. J., Visser, R. G. F, and Jacobsen, E.    (2004). The R3 resistance to Phytophthora infestans in potato is    conferred by two closely linked R genes with distinct specificities.    MPMI 17 (4), 428-435.

In one embodiment, the activity of the Rpi-blb2 is increased accordingto the present invention, e.g. the polynucleotide of the invention'sexpression is increased and the expression of at least one nucleic acidmolecule is increased encoding Rpi-blb, Rpi-ABPT1, Rpi-blb3, Rpi-mcd R1,R-ber, Rpi1, R2, R3a, R3b, R6, R7, Ph-1, Ph-2 and/or Ph-3 whereby thenucleic acid molecule is selected from the group consisting of:

-   a) nucleic acid molecule encoding at least a mature form of at least    -   a Rpi-blb (or RB- or Sbu1-) polypeptide, preferably as encoded        by the sequence shown in GenBank Accession no.: AY336128 [gi:        32693280];    -   a R1 polypeptide, preferably as encoded by the sequence shown in        GenBank Accession no.: AF447489 [gi 9117432422];    -   a Rpi-blb3, Rpi-ABPT1 and/or Rpi-mcd polypeptide, preferably        encoded by the sequence shown in or derivable by the information        given in Park, T. H., Van der Vossen, E., Vleeshouwers, V. G. A.        A., Tan, A., Visser, R. G. F. and Van Eck, H. J. 2004. Major        resistance genes for tuber and leaf resistance to Phytophthora        infestans in potato: An outline of a PhD project. Crop        Functional Genomics 2004, July 2004, Jeju, Korea, page 93;    -   a R3a and/or R3b polypeptide, preferably encoded by the sequence        shown in or derivable by the information given in Huang, S.,        Vleeshouwers, V. G. A. A., Werij, J. S., Hutten, R. C. B., Van        Eck, H. J., Visser, R. G. F, and Jacobsen, E. (2004). The R3        resistance to Phytophthora infestans in potato is conferred by        two closely linked R genes with distinct specificities. MPMI 17        (4), 428-435 and/or    -   a pathogen, preferably P. infestans, resistance conferring        protein mapped and characterized as described, e.g. as for    -   for Rpi1 in Kuhl, J. C., Hanneman, R. E., and Havey, M.        J., (2001) Characterization and mapping of Rpi1, a late blight        resistance locus from diploid (1EBN) Mexican Solanum        pinnatisectum. Molecular genet. Genomics 265: 977-985;    -   for R-ber in Ewing, E. E., Simko, I., Smart, C. D.,        Bonierbale, M. W., Mizubuti, E. S. G., May, G. D., and Fry, W.        E., (2000) Genetic mapping from field tests of qualitative and        quantitative resistance to Phytophthora infestans in a        population derived from Solanum tuberosum and Solanum        berthaultii. Molecular breeding 6:25-36;    -   for R2 in L1, X., vanEck, H. J., vanderVoort, J. N. A. M.,        Huigen, D. J., Stam, P., and Jacobsen, E. (1998) Autotetraploids        and genetic mapping using common AFLP markers: the R2 allele        conferring resistance to Phytophthora infestans mapped on potato        chromosome 4. Theoretical and Applied Genetics 96 (8):        1121-1128;    -   for R3, R6, R7 in Elkharbotly, A., Palominosanchez, C.,        Salamini, F., Jacobsen, E., and Gebhardt, C. (1996) R6 and R7        alleles of potato conferring race-specific resistance to        Phytophthora infestans (Mont) de Bary identified genetic loci        clustering with the R3 locus on chromosome XI. Theoretical and        Applied. Genetics 92 (7): 880-884;    -   for Ph-1 in Bonde and Murphy (1952) Main Agric. Exp. Stn. Bull.        No 497; or    -   for Ph-2 in Moreau, P., Thoquet, P., Olivier, J., Laterrot, H.,        and Grimsley, N. H. (1998) Genetic mapping of Ph-2, a single        locus controlling partial resistance to Phytophthora infestans        in tomato. Molecular Plant Microbe Interactions 11 (4): 259-269;        and/or    -   for Ph-3 in Chunwongse, J., Chunwongse, C., Black, L., and        Hanson, P. (2002) Molecular mapping of the Ph-3 gene for late        blight resistance in tomato. Journal of Horticultural Science &        Biotechnology 77 (3): 281-286;    -   or a pathogen resistance conferring polypeptide, preferably P.        infestans resistance conferring polypeptide derivable from said        publications;-   b) nucleic acid molecule the nucleotide sequence of which is    degenerate as a result of the genetic code to a nucleotide sequence    of (a);-   c) nucleic acid molecule encoding a polypeptide derived from the    polypeptide encoded by a polynucleotide of (a) or (b) by way of    substitution, deletion and/or addition of one or several amino acids    of the amino acid sequence of the polypeptide encoded by a    polynucleotide of (a) or (b);-   d) nucleic acid molecule encoding a polypeptide the sequence of    which has an identity of 70% or more to the amino acid sequence of    the polypeptide encoded by a nucleic acid molecule of (a);-   e) nucleic acid molecules comprising a fragment or a epitope-bearing    portion of a polypeptide encoded by a nucleic acid molecule of any    one of (a) to (d);-   f) nucleic acid molecule encoding a fragment beginning with amino    acid: 1, 30, 50, 100, 200, 500 or 1000, and stopping with amino acid    1267, 1000, 500, 300, 200, 50, 30, or 1 of a polypeptide encoded by    any one of (a) to (e) and with one of said activities;-   g) nucleic acid molecule comprising at least 20 nucleotides of a    polynucleotide of any one of (a) or (b);-   h) nucleic acid molecule encoding a polypeptide being recognized by    a monoclonal antibody that have been raised against a polypeptide    encoded by a nucleic acid molecule of any one of (a) to (f);-   i) nucleic acid molecule obtainable by screening an appropriate    library under stringent conditions with a probe having the sequence    of the nucleic acid molecule of any one of (a) to (h) or of a    fragment thereof of at least 20, preferable 30 or more nucleotides;    and-   j) nucleic acid molecule the complementary strand of which    hybridises under stringent conditions with a nucleic acid molecule    of any one of (a) or (i);    or the complementary strand of any one of (a) to (j).

Accordingly, the method of present invention confers resistance of oneof said plants, plant tissue or plant cell of the present invention to aplant pathogen of a phylum Oomycetes, preferably to a pathogen of theorder Pythiales or Peronosperales, more preferred to the familyPythiaceae or Peronosporaceae, more preferred of the genus Phytophthoraor Bremia or Peronospera or Plasmopara, most preferred wherein thepathogen is of the species Phytophthora parasitica var. nicotianae(causing, amongst others, black shank in tobacco), Phytophthora sojae(causing Phytophthora root rot in soybean), Phytophthora capsici(causing rots in pepper and cucurbits and tomato), Phytophthoraerythroseptica (causing Pink rot in potato), Plasmopara viticola(causing grapevine downy mildew), Bremia lactuca (causing downy mildewin lettuce) or Peronospora tabaci (causing blue mould in tobacco).

The activity of Rpi-blb2 in a plant, a plant cell, a plant tissue, aplant organ or part thereof according to the present invention can beincreased, generated or stimulated via methods which are well known to aperson skilled in the art and e.g. are described in Sambrook et al.,Cold Spring Harbor Laboratory Press, NY, 1989.

Thus, in a preferred embodiment, the present invention relates to themethod of the invention, wherein the expression is a de novo expression.

The term “de novo-Expression” in a cell, a tissue or in an organism orin a part thereof as understood herein relates to the expression of agene product after a previous non-detectability of said gene product oran activity of said gene product, in particular of a correspondingpolypeptide or polynucleotide in a cell, a tissue or in an organism orin a part thereof. Preferred is that the gene encoding a polypeptide ora polynucleotide in a cell, a tissue or in an organism or in partsthereof and which should be de novo-expressed is not present in thegenome in a cell, a tissue or in an organism or in parts thereof. If theexpression of a gene product can not be detected in a cell, a tissue orin an organism or in parts thereof, it is generally assumed that noexpression occurs in a cell, a tissue or in an organism or in partsthereof. Accordingly, if the activity can not be detected, it isgenerally assumed that no corresponding activity exists. A personskilled in the art, however, knows that the detection methods and meansdevelop to higher sensitivity. Thus, in a preferred embodiment, the term“de novo-Expression” relates to a novel or additional expression insystems, where the level of activity, e.g. due to a low expression levelor the expression of an (nearly) inactive gene product is too low toconfer any resistance to a plant pathogen, in particular to P.infestans. A comparison of a knock out strain and a low and/orhigh-expression strain-phenotype can show, whether any difference inresistance to any of the herein mentioned pathogens is observable.

Accordingly, in another embodiment of the present invention, theendogenous activity of a Rpi-blb2 and/or a further resistance protein isincreased.

The level of expression in a cell can be increased by methods known to aperson skilled in the art. Several techniques are described herein, e.g.the transgenic expression of the polynucleotide or polypeptide of thepresent invention. The polynucleotide or polypeptide can be of foreignorigin. Preferred is that a polynucleotide of the same genetic origin asthe host cell, plant cell, plant tissue, or plant is introduced.

The activity, in particular an endogenous activity but also the activityof a transgenic expressed Rpi-blb2 can be increased by several methods.Accordingly, in a preferred embodiment, the activity of the resistanceproteins described herein is increased by one or more of the followingsteps

-   a) stabilizing the resistance protein;-   b) stabilizing the resistance protein encoding mRNA;-   c) increasing the specific activity of the resistance protein;-   d) expressing or increasing the expression of a homologous or    artificial transcription factor for resistance expression;-   e) stimulate resistance protein activity through exogenous inducing    factors;-   f) expressing a transgenic resistance gene; and/or-   g) increasing the copy number of the resistance-encoding gene.

In general an activity in an organism, in particular in a plant cell, aplant, or a plant tissue can be increased by increasing the amount ofthe specific protein, i.e. of the resistance protein, in said organism.“Amount of protein” is understood as meaning the amount of apolypeptide, preferably Rpi-blb2, in an organism, a tissue, a cell or acell compartment. “Increase” of the amount of protein means thequantitative increase of the amount of a protein in an organism, atissue, a cell or a cell compartment—for example by one of the methodsdescribed herein below—in comparison with the wild type of the samegenus and species, to which this method had not been applied, underotherwise identical conditions (such as, for example, cultureconditions, plant age and the like). The increase amounts to at least10%, preferably at least 20% or at least 50%, especially preferably atleast 70% or 90%, very especially preferably at least 100%, mostpreferably at least 200% or more.

“Increase” of the activity is understood as meaning the increase of thetotal activity of a protein in an organism, a tissue, a cell or a cellcompartment in comparison with the wild type of the same genus andspecies, to which this method had not been applied, under otherwiseidentical conditions (such as, for example, culture conditions, plantage and the like). The increase amounts to at least 10%, preferably atleast 20% or at least 50%, especially preferably at least 70% or 90%,very especially preferably at least 100%, most preferably at least 200%or more.

In this context, the efficacy of the pathogen resistance can deviateboth downward or upward in comparison with a value obtained whenincreasing one of the Rpi-blb2 proteins as shown in SEQ ID NO: 2 or 4.Preferred functional equivalents are those in which the efficacy of thepathogen resistance—measured, for example, by the penetration efficacyof a pathogen or as described herein—differs by not more than 50%,preferably 25%, especially preferably 10% from a comparative valueobtained by reducing a Rpi-blb2 protein as shown in SEQ ID NO: 2 or 4.Especially preferred are those sequences where the increase increasesthe efficacy of pathogen resistance quantitatively by more than 50%,preferably 100%, especially preferably 500%, very especially preferably1000% based on a comparative value obtained by reducing one of theRpi-blb2 proteins as shown in SEQ ID NO: 2 or 4.

Any comparison is preferably carried out under analogous conditions.“Analogous conditions” means that all conditions such as, for example,culture or growing conditions, assay conditions (such as buffer,temperature, substrates, pathogen concentration and the like) are keptidentical between the experiments to be compared and that the set-upsdiffer only by the sequence of the Rpi-blb2 polypeptides to be compared,their organism of origin and, if appropriate, the pathogen. Whenchoosing the pathogen, each comparison requires that the pathogen bechosen which is most similar to the other equivalent, taking intoconsideration the species specificity.

Due to the increased Rpi-blb2 activity, the resistance of a plant or apart thereof is increased. In a preferred embodiment, the method of thepresent invention results in reduction in the sporulation index of atleast 30% after infection with P. infestans compared to a wild type,more preferred is a reduction of 50%, even more preferred are 70%, evenmore preferred are more than 80%, more preferred are 85% and 90%. Mostpreferred is 95% or more.

Accordingly, the present invention also relates to said polynucleotideof the invention, as defined above encoding a Rpi-blb2 proteincomprising a nucleic acid molecule selected from the group consistingof:

-   a) nucleic acid molecules encoding at least the mature form of the    polypeptide depicted in SEQ ID NO: 2 or 4;-   b) nucleic acid molecules comprising the coding sequence as depicted    in SEQ ID NO: 1 or 3 or 5 or 6 or encoding at least the mature form    of the polypeptide;-   c) nucleic acid molecules the nucleotide sequence of which is    degenerate as a result of the genetic code to a nucleotide sequence    of (a) or (b);-   d) nucleic acid molecules encoding a polypeptide derived from the    polypeptide encoded by a polynucleotide of (a) to (c) by way of    substitution, deletion and/or addition of one or several amino acids    of the amino acid sequence of the polypeptide encoded by a    polynucleotide of (a) to (c);-   e) nucleic acid molecules encoding a polypeptide the sequence of    which has an identity of 70% or more to the amino acid sequence of    the polypeptide encoded by a nucleic acid molecule of (a) or (b);-   f) nucleic acid molecules comprising a fragment or a epitope-bearing    portion of a polypeptide encoded by a nucleic acid molecule of any    one of (a) to (e);-   g) nucleic acid molecules comprising a polynucleotide having a    sequence of a nucleic acid molecule amplified from a nucleic acid    library using the primers as listed in Tab. 3b, preferably ARF1F or    ARF1R;-   h) nucleic acid molecules encoding polypeptide fragment beginning    with amino acid: 1, 30, 50, 100, 200, 300, 500, or 1000 and stopping    with, amino acid 1267, 1000, 500, 300, 200, 50, 30, or 1 of a    polypeptide encoded by any one of (a) to (g);-   i) nucleic acid molecules comprising at least 20 nucleotides of a    polynucleotide of any one of (a) or (d);-   j) nucleic acid molecules encoding a polypeptide being recognized by    a monoclonal antibodies that have been raised against a polypeptide    encoded by a nucleic acid molecule of any one of (a) to (h);-   k) nucleic acid molecules obtainable by screening an appropriate    library under stringent conditions with a probe having the sequence    of the nucleic acid molecule of any one of (a) to (j) or of a    fragment thereof of at least 15, preferable 30, 60, 90 or more    nucleotides; and-   l) nucleic acid molecules the complementary strand of which    hybridises under stringent conditions with a nucleic acid molecule    of any one of (a) or (k);    or the complementary strand of any one of (a) to (l);    or encoding a polypeptide encoded by a segment of chromosome 6 or of    linkage group 6 of Solanum bulbocastanum which co-segregates with a    marker selected from table 3a or 3b and which mediates resistance to    plant pathogens, preferably of the phylum Oomycetes;

In one embodiment, the polynucleotide of the invention does not consistof the sequence depicted in Seq. ID NO.: 7 and/or 9 and/or does notconsist of the sequence of a nucleic acid molecule encoding a proteindepicted in Seq. ID NO.: 8 and/or 10.

In one embodiment, the polynucleotide of the present invention does notconsist of the sequence of a nucleic acid molecule of Mi1.1 or Mi1.2and/or of a nucleic acid molecule encoding a Mi1.1 or Mi1.2 protein.

Thus, in one embodiment, the polynucleotide of the present invention maynot consist of the sequences shown in Rossi et al. 1998, PNAS USA95:9750-9754, Milligan et al., 1998. Plant Cell 10:1307-1319; and/or WO9806750.

In an further embodiment, the polynucleotide of the present invention isderived or isolated from the genome of a organism selected from thegroup consisting of Menyanthaceae, Solanaceae, Sclerophylacaceae,Duckeodendraceae, Goetzeaceae, Convolvulaceae, Cuscutaceae,Polemoniaceae, and Hydrophyllaceae according to the Systema Naturae2000, Brands, S. J., Amsterdam or has its origin thereof, morepreferably it is selected from the group consisting of Atropa,Browallia, Brunfelsia, Capsicum, Cestrum, Cyphomandra, Datura, Fabiana,Frariciscea, Hyoscyamus, Lycium, Mandragora, Nicandra, Nicotiana,Petunia, Physalis, Schizanthus and Solanum according to the SystemaNaturae 2000, Brands, S. J., Amsterdam or has its origin thereof, evenmore preferred is a selection out of the group consisting of Solanaceaefamily, preferably S. bulbocastanum, potato (S. tuberosum), tomato (S.lycopersicum), petunia, tree tomato (S. betaceum), pear melon (S.muricatum) and eggplant (S. melongena). Even more preferred are tomatoor potato or S. bulbocastanum as source for the polynucleotide of thepresent invention. Most preferred is S. bulbocastanum as source.

Rpi-blb2 has been isolated from S. tuberosum material derived form ABPT.Thus, from taxonomic perspective the Rpi-blb2 described is also S.tuberosum-derived. However, the gene was present on an introgressionfragment presumably derived from S. bulbocastanum. A lot of S. tuberosumvarieties contain introgression fragments of related Solanum species,but still are S. tuberosum. Therefore, S. tuberosum can according to thetaxonomical system also be a source for the polynucleotide of thepresent invention, in particular ABPT-derived S. tuberosum, as well asother varieties of other Solanum species varieties derived in a similarway.

Accordingly, in another embodiment the polynucleotide of the presentinvention is derived from S. tuberosum.

A polynucleotide of the present invention, e.g., a nucleic acid moleculehaving a nucleotide sequence of Seq ID NO: 1 or 3 or 5 or 6, or aportion thereof, can be isolated using standard molecular biologytechniques and the sequence information provided herein. For example,Rpi-blb2 cDNA can be isolated from a library using all or portion of oneof the sequences of the polynucleotide of the present invention as ahybridisation probe and standard hybridisation techniques (e.g., asdescribed in Sambrook et al., Molecular Cloning: A Laboratory Manual.2^(nd), ed., Cold Spring Harbor Laboratory, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989). Moreover, apolynucleotide encompassing all or a portion of one of the sequences ofthe polynucleotide of the present invention can be isolated by thepolymerase chain reaction using oligonucleotide primers designed basedupon this sequence (e.g., a nucleic acid molecule encompassing all or aportion of one of the sequences of polynucleotide of the presentinvention). For example, mRNA can be isolated from cells, e.g. S.bulbocastanum or another plant (e.g., by the guanidinium-thiocyanateextraction procedure of Chirgwin et al. (1979) Biochemistry 18:5294-5299) and cDNA can be prepared using reverse transcriptase (e.g.,Moloney MLV reverse transcriptase, available from Gibco/BRL, Bethesda,Md.; or AMV reverse transcriptase, available from Seikagaku America,Inc., St. Petersburg, Fla.). Synthetic oligonucleotide primers forpolymerase chain reaction amplification can be designed based upon oneof the nucleotide sequences shown in SEQ ID No: 1 or 3 or 5 or 6. Apolynucleotide of the invention can be amplified using cDNA or,alternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The polynucleotide so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to a Rpi-blb2 nucleotidesequence can be prepared by standard synthetic techniques, e.g., usingan automated DNA synthesizer.

In an embodiment of the present invention the Rpi-blb 2 protein isencoded by a segment of chromosome 6 or linkage group 6 of Solanumbulbocastanum or S. tuberosum.

Further the present invention comprises a segment of chromosome 6 orlinkage group 6 of S. bulbocastanum or S. tuberosum. In one preferredembodiment in the method of the present invention the Rpi-blb2 proteinexpressed is encoded by a polynucleotide comprising a segment ofchromosome 6 or linkage group 6 of S. bulbocastanum. Preferably saidsegment a group comprises further cis acting element, e.g. promoters,enhancers, binding sites etc. or trans acting elements, like cofactors,activators or other resistance proteins, which confer an increasedresistance. Genomic fragments comprising the Rpi-blb2 gene and furtherregulatory elements are depicted in Seq. ID NO.: 5 and 6.

A person skilled in the art knows how to obtain a chromosome segment,e.g. by cloning chromosome fragments into BACs, as for example Song,2003, PNAS 100 (16), 9128 or as described herein and in the referencescited herein.

Accordingly, in a further embodiment, the polynucleotide of the presentinvention or a polynucleotide encoding the Rpi-blb2 proteinco-segregates with a marker selected from table 3a or comprises areplication site or hybridisation site for said marker. As described indetail in the examples, the resistance to P. infestans could be mappedwith the markers depicted in table 3a or 3b. As closer a marker islocalized to a gene, as higher is the percentage of lines, i.e.offspring clones, in which the gene co-segregates with said marker.Therefore in a preferred embodiment, the polynucleotide of the presentinvention co-segregates with the Marker E40M58, CT119 and/or CT216.

In a further embodiment, the present invention relates to a method formaking a recombinant vector comprising inserting the polynucleotide ofthe present invention into a vector or inserting said polynucleotide anda further resistance protein into a vector.

Accordingly, in one further embodiment, the present invention relates toa vector containing the polynucleotide of the present invention or saidpolynucleotide and a further resistance gene produced by the method ofthe present invention.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting a polynucleotide to which it has been linked.One type of vector is a “plasmid”, which refers to a circular doublestranded DNA loop into which additional DNA segments can be ligated.Another type of vector is a viral vector, wherein additional DNA or RNAsegments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively linked.Such vectors are referred to herein as “expression vectors”. In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” can be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The present invention also relates to cosmids, viruses, bacteriophagesand other vectors used conventionally in genetic engineering thatcontain a nucleic acid molecule according to the invention. Methodswhich are well known to those skilled in the art can be used toconstruct various plasmids and vectors; see, for example, the techniquesdescribed in Sambrook, Molecular Cloning A Laboratory Manual, ColdSpring Harbor Laboratory (1989) N.Y. and Ausubel, Current Protocols inMolecular Biology, Green Publishing Associates and Wiley Interscience,N.Y. (1989). Alternatively, the nucleic acid molecules and vectors ofthe invention can be reconstituted into liposomes for delivery to targetcells.

In another embodiment, the vector of the present invention or the methodof the present invention is characterized therein, that thepolynucleotide encoding Rpi-blb2 protein or a further resistance proteinis operatively linked to expression control sequences and/or a linked toa nucleic acid sequence encoding a transgenic expression regulatingsignal allowing expression in prokaryotic or eukaryotic host cells.

In a preferred embodiment, the present invention relates to a vector ofthe present invention or the method of the present invention in whichthe polynucleotide encoding Rpi-blb2 protein and/or the furtherresistance protein is operatively linked to expression control sequencesof the same species origin as the polynucleotide encoding Rpi-blb2protein and/or the further resistance protein.

In the case that a nucleic acid molecule according to the invention isexpressed in a cell it is in principle possible to modify the codingsequence in such a way that the protein is located in any desiredcompartment of the plant cell. These include the nucleus, endoplasmaticreticulum, the vacuole, the mitochondria, the plastids like amyloplasts,chloroplasts, chromoplasts, the apoplast, the cytoplasm, extracellularspace, oil bodies, peroxisomes and other compartments of plant cells(for review see Kermode, Crit. Rev. Plant Sci. 15, 4 (1996), 285-423 andreferences cited therein). The polynucleotide can then operatively befused to an appropriate polynucleotide, e.g., a vector, encoding asignal for the transport into the desirable compartment.

In another preferred embodiment of the present invention relates to avector in which the polynucleotide of the present invention isoperatively linked to expression control sequences allowing expressionin prokaryotic or eukaryotic host cells. The nature of such controlsequences differs depending upon the host organism. In prokaryotes,control sequences generally include promoter, ribosomal binding site,and terminators. In eukaryotes, generally control sequences includepromoters, terminators and, in some instances, enhancers,transactivators; or transcription factors.

The term “control sequence” is intended to include, at a minimum,components the presence of which are necessary for expression, and mayalso include additional advantageous components.

The term “operatively linked” refers to a juxtaposition wherein thecomponents so described are in a relationship permitting them tofunction in their intended manner. A control sequence “operativelylinked” to a coding sequence is ligated in such a way that expression ofthe coding sequence is achieved under conditions compatible with thecontrol sequences. In case the control sequence is a promoter, it isobvious for a skilled person that double-stranded nucleic acid is used.

Operable linkage is to be understood as meaning, for example, thesequential arrangement of a promoter with the nucleic acid sequence tobe expressed and, if appropriate, further regulatory elements such as,for example, a terminator in such a way that each of the regulatoryelements can fulfil its function when the nucleic acid sequence isexpressed recombinantly, depending on the arrangement of the nucleicacid sequences in relation to sense or antisense RNA. To this end,direct linkage in the chemical sense is not necessarily required.Genetic control sequences such as, for example, enhancer sequences, canalso exert their function on the target sequence from positions whichare further away, or indeed from other DNA molecules. Preferredarrangements are those in which the nucleic acid sequence to beexpressed recombinantly is positioned behind the sequence acting aspromoter, so that the two sequences are linked covalently to each other.The distance between the promoter sequence and the nucleic acid sequenceto be expressed recombinantly is preferably less than 200 base pairs,especially preferably less than 100 base pairs, very especiallypreferably less than 50 base pairs.

Operable linkage, and an expression cassette, can be generated by meansof customary recombination and cloning techniques as are described, forexample, in Maniatis T, Fritsch E F and Sambrook J (1989) MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor (NY), in Silhavy T J, Berman M L and Enquist L W (1984)Experiments with Gene Fusions, Cold Spring Harbor Laboratory, ColdSpring Harbor (N.Y.), in Ausubel F M et al. (1987) Current Protocols inMolecular Biology, Greene Publishing Assoc. and Wiley Interscience andin Gelvin et al. (1990) In: Plant Molecular Biology Manual. However,further sequences which, for example, act as a linker with specificcleavage sites for restriction enzymes, or as a signal peptide, may alsobe positioned between the two sequences. The insertion of sequences mayalso lead to the expression of fusion proteins. Preferably, theexpression cassette, consisting of a linkage of promoter and nucleicacid sequence to be expressed, can exist in a vector-integrated form andbe inserted into a plant genome, for example by transformation.

Such regulatory sequences are described, for example, in Goeddel; GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) or see: Gruber and Crosby, in: Methods in PlantMolecular Biology and Biotechnology, CRC Press, Boca Raton, Fla., eds.:Glick and Thompson, Chapter 7, 89-108 including the references therein.Regulatory sequences include those which direct constitutive expressionof a nucleotide sequence in many types of host cell and those whichdirect expression of the nucleotide sequence only in certain host cellsor under certain conditions. It will be appreciated by those skilled inthe art that the design of the expression vector can depend on suchfactors as the choice of the host cell to be transformed, the level ofexpression of protein desired, etc. The expression vectors of theinvention can be introduced into host cells to thereby produce proteinsor peptides, including fusion proteins or peptides, encoded bypolynucleotides as described herein.

The recombinant expression vectors of the invention can be designed forexpression of said resistance proteins, preferably Rpi-blb2, inprokaryotic or eukaryotic cells. For example, genes encoding thepolynucleotide of the invention can be expressed in bacterial cells suchas E. coli, C. glutamicum, Agrobacterium tumefaciens, insect cells(using baculovirus expression vectors), yeast and other fungal cells(see Romanos, (1992), Yeast 8: 423-488; van den Hondel, (1991) J. W.Bennet & L. L. Lasure, eds., p. 396-428: Academic Press: San Diego; andvan den Hondel, (1991) in: Applied Molecular Genetics of Fungi, Peberdy,eds., p. 1-28, Cambridge University Press: Cambridge), algae (Falciatoreet al., 1999, Marine Biotechnology. 1, 3:239-251), and multicellularplant cells (see Schmidt, R. (1988), Plant Cell Rep.: 583-586); PlantMolecular Biology and Biotechnology, C Press, Boca Raton, Fla., chapter6/7, S.71-119 (1993); F. F. White, B. Jenes et al., Techniques for GeneTransfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization,eds.: Kung und R. Wu, Academic Press (1993), 128-43; Potrykus, Annu.Rev. Plant Physiol. Plant Molec. Biol. 42 (1991), 205-225 (andreferences cited therein) or mammalian cells. Suitable host cells arediscussed further in Goeddel, Gene Expression Technology: Methods inEnzymology 185, Academic Press, San Diego, Calif. (1990). Alternatively,the recombinant expression vector can be transcribed and translated invitro, for example using T7 promoter regulatory sequences and T7polymerase.

Expression of proteins in prokaryotes is most often carried out withvectors containing constitutive or inducible promoters directing theexpression of either fusion or non-fusion proteins. Fusion vectors add anumber of amino acids to a protein encoded therein, usually to the aminoterminus of the recombinant protein but also to the C-terminus or fusedwithin suitable regions in the proteins. Such fusion vectors typicallyserve three purposes: 1) to increase expression of recombinant protein;2) to increase the solubility of the recombinant protein; and 3) to aidin the purification of the recombinant protein by acting as a ligand inaffinity purification. Further, the fusion vector can also encode foradditional proteins, which expression supports an increase of theactivity of Rpi-blb2 or of the resistance of a plant against plantpathogens, e.g. other resistance proteins. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin, and enterokinase.

Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New EnglandBiolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.).

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann (1988) Gene 69:301-315) and pET 11d (Studier et al.,Gene Expression Technology: Methods in Enzymology 185, Academic Press,San Diego, Calif. (1990) 60-89.

One strategy to maximize recombinant protein expression is to expressthe protein in a host bacterium with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman, S., GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 119-128). Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in the bacterium chosen for expression, such asE. coli or C. glutamicum (Wada et al. (1992) Nucleic Acids Res.20:2111-2118). Such alteration of nucleic acid sequences of theinvention can be carried out by standard DNA synthesis techniques.

Further, the vector can be a yeast expression vector. Examples ofvectors for expression in yeast S. cerivisae include pYepSec1 (Baldari,et al., (1987) Embo J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982)Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123), andpYES2 (Invitrogen Corporation, San Diego, Calif.).

Preferably, the polynucleotide of the present invention or describedherein is operatively linked to a plant expression control sequence,e.g. an expression cassettes. A plant expression cassette preferablycontains regulatory sequences capable to drive gene expression in plantscells and which are operatively linked so that each sequence can fulfilits function such as termination of transcription such aspolyadenylation signals. Preferred polyadenylation signals are thoseoriginating from Agrobacterium tumefaciens t-DNA such as the gene 3known as octopine synthase of the Ti-plasmid pTiACH5 (Gielen et al.,EMBO J. 3 (1984), 835 ff) or functional equivalents thereof but also allother terminators functionally active in plants are suitable.

As plant gene expression is very often not limited on transcriptionallevels as plant expression cassette preferably contains otheroperatively linked sequences like translational enhancers such as theoverdrive-sequence containing the 5′-untranslated leader sequence fromtobacco mosaic virus enhancing the protein per RNA ratio (Gallie et al1987, Nucl. Acids Research 15:8693-8711).

Accordingly, the polynucleotide described herein can be operativelylinked to an appropriate promoter conferring gene expression in atimely, cell or tissue specific manner. Preferred are promoters drivingconstitutitive expression (Benfey et al., EMBO J. 8 (1989) 2195-2202)like those derived from plant viruses like the 35S CAMV (Franck et al.,Cell 21(1980) 285-294), the 19S CaMV (see also U.S. Pat. No. 5,352,605and WO8402913) or plant promoters like those from Rubisco small subunitdescribed in U.S. Pat. No. 4,962,028.

The term plant-specific promoters is understood as meaning, inprinciple, any promoter which is capable of governing the expression ofgenes, in particular foreign genes, in plants or plant parts, plantcells, plant tissues or plant cultures. In this context, expression canbe, for example, constitutive, inducible, or development-dependent.

The following are preferred:

a) Constitutive Promoters

Preferred vectors are those which make possible constitutive expressionin plants (Benfey et al. (1989) EMBO J. 8:2195-2202). “Constitutive”promoter is understood as meaning those promoters which ensureexpression in a large number of, preferably all, tissues over asubstantial period of plant development, preferably at all stages ofplant development. In particular a plant promoter or a promoter derivedfrom a plant virus are preferably used. Particularly preferred is thepromoter of the CaMV cauliflower mosaic virus 35S transcript (Franck etal. (1980) Cell 21:285-294; Odell et al. (1985) Nature 313:810-812;Shewmaker et al. (1985) Virology 140:281-288; Gardner et al. (1986)Plant Mol Biol 6:221-228) or the 19S CaMV promoter (U.S. Pat. No.5,352,605; WO 84/02913; Benfey et al. (1989) EMBO J. 8:2195-2202).Another suitable constitutive promoter is the “Rubisco small subunit(SSU)” promoter (U.S. Pat. No. 4,962,028), the leguminB promoter(GenBank Acc. No. X03677), the Agrobacterium nopaline synthase promoter,the TR dual promoter, the Agrobacterium OCS (octopine synthase)promoter, the ubiquitin promoter (Holtorf S et al. (1995) Plant Mol Biol29:637-649), the ubiquitin 1 promoter (Christensen et al. (1992) PlantMol Biol 18:675-689; Bruce et al. (1989) Proc Natl Acad Sci USA86:9692-9696), the Smas promoter, the cinnamyl alcohol dehydrogenasepromoter (U.S. Pat. No. 5,683,439), the promoters of the vacuolar ATPasesubunits or the promoter of a proline-rich protein from wheat (WO91/13991), and further promoters of genes whose constitutive expressionin plants is known to the skilled worker.

b) Tissue-specific Promoters

Preferred are furthermore promoters with specificity for the anthers,ovaries, flowers, leaves, stems, roots, and seeds.

Seed-specific Promoters

such as, for example, the phaseolin promoter (U.S. Pat. No. 5,504,200;Bustos M M et al. (1989) Plant Cell 1(9):839-53), the 2S albumin genepromoter (Joseffson L G et al. (1987) J Biol Chem 262:12196-12201), thelegumin promoter (Shirsat A et al. (1989) Mol Gen Genet 215(2):326-331), the USP (unknown seed protein) promoter (Bäumlein H et al.(1991) Mol Gen Genet 225(3):459-67), the napin gene promoter (U.S. Pat.No. 5,608,152; Stalberg K et al. (1996) L Planta 199:515-519), thesucrose binding protein promoter (WO 00/26388) or the legumin B4promoter (LeB4; Bäumlein H et al. (1991) Mol Gen Genet 225: 121-128;Baeumlein et al. (1992) Plant Journal 2(2):233-9; Fiedler U et al.(1995) Biotechnology (NY) 13(10):1090f), the Arabidopsis oleosinpromoter (WO 98/45461), the Brassica Bce4 promoter (WO 91/13980).Further suitable seed-specific promoters are those of the genes encodingthe high-molecular-weight glutenin (HMWG), gliadin, branching enzyme,ADP glucose pyrophosphatase (AGPase) or starch synthase. Furthermorepreferred are promoters which permit seed-specific expression inmonocots such as maize, barley, wheat, rye, rice and the like. Thefollowing can be employed advantageously: the promoter of the lpt2 orlpt1 gene (WO 95/15389, WO 95/23230) or the promoters described in WO99/16890 (promoters of the hordein gene, the glutelin gene, the oryzingene, the prolamin gene, the gliadin gene, the glutelin gene, the zeingene, the kasirin gene or the secalin gene).

Tuber-, storage-root-, or root-specific promoters such as, for example,the patatin promoter class I (B33), the potato cathepsin D inhibitorpromoter.

Leaf-specific Promoters

such as the potato cytosolic FBPase promoter (WO 97/05900), the Rubisco(ribulose-1,5-bisphosphate carboxylase) SSU (small subunit) promoter orthe ST-LSI promoter from potato (Stockhaus et al. (1989) EMBO J.8:2445-2451). Very especially preferred are epidermis-specific promoterssuch as, for example, the OXLP gene (oxalate-oxidase-like protein)promoter (Wei et al. (1998) Plant Mol. Biol. 36:101-112).

Flower-specific Promoters

such as, for example, the phytoene synthase promoter (WO 92/16635) orthe promoter of the P-rr gene (WO 98/22593).

Anther-specific Promoters

such as the 5126 promoter (U.S. Pat. No. 5,689,049, U.S. Pat. No.5,689,051), the glob-I promoter and the γ-zein promoter.

c) Chemically Inducible Promoters

The expression cassettes can also comprise a chemically induciblepromoter (review article: Gatz et al. (1997) Annu Rev Plant PhysiolPlant Mol Biol 48:89-108), by which the expression of the exogenous genein the plant at a particular point in time can be controlled. Suchpromoters such as, for example, the PRP1 promoter (Ward et al. (1993)Plant Mol Biol 22:361-366), a salicylic-acid-inducible promoter (WO95/19443), a benzenesulfonamide-inducible promoter (EP 0 388 186), atetracycline-inducible promoter (Gatz et al. (1992) Plant J 2:397-404),an abscisic-acid-inducible promoter (EP 0 335 528) or an ethanol- orcyclohexanone-inducible promoter (WO 93/21334) can likewise be used.

d) Stress- or Pathogen-inducible Promoters

Further preferred promoters are those which are induced by biotic orabiotic stress such as, for example, the pathogen-inducible promoter ofthe PRP1 gene (Ward et al. (1993) Plant Mol Biol 22:361-366), the tomatohigh-temperature-inducible hsp70 or hsp80 promoter (U.S. Pat. No.5,187,267), the potato low-temperature-inducible alpha-amylase promoter(WO 96/12814), the light-inducible PPDK promoter, or thewounding-induced pinII promoter (EP375091).

Pathogen-inducible promoters encompass those of genes which are inducedas a consequence of infection by pathogens, such as, for example, genesof PR proteins, SAR proteins, β-1,3-glucanase, chitinase and the like(for example Redolfi et al. (1983) Neth J Plant Pathol 89:245-254;Uknes, et al. (1992) The Plant Cell 4:645-656; Van Loon (1985) Plant MolVirol 4:111-116; Marineau et al. (1987) Plant Mol Biol 9:335-342; Mattonet al. (1987) Molecular Plant-Microbe Interactions 2:325-342; Somssichet al. (1986) Proc Natl Acad Sci USA 83:2427-2430; Somssich et al.(1988) Mol Gen Genetics 2:93-98; Chen et al. (1996) Plant J 10:955-966;Zhang and Sing (1994) Proc Natl Acad Sci USA 91:2507-2511; Warner, etal. (1993) Plant J 3:191-201; Siebertz et al. (1989) Plant Cell1:961-968(1989).

Also encompassed are wounding-inducible promoters such as that of thepinII gene (Ryan (1990) Ann Rev Phytopath 28:425-449; Duan et al. (1996)Nat Biotech 14:494-498), of the wun1 and wun2 gene (U.S. Pat. No.5,428,148), of the win1 and win2 gene (Stanford et al. (1989) Mol GenGenet 215:200-208), of systemin (McGurl et al. (1992) Science225:1570-1573), of the WIP1 gene (Rohmeier et al. (1993) Plant Mol Biol22:783-792; Eckelkamp et al. (1993) FEBS Letters 323:73-76), of the MPIgene (Corderok et al. (1994) The Plant J 6(2):141-150) and the like.

e) Development-dependent Promoters

Further suitable promoters are, for example, fruit-maturation-specificpromoters such as, for example, the tomato fruit-maturation-specificpromoter (WO 94/21794, EP 409 625). Development-dependent promoterscomprise partly the tissue-specific promoters, since individual tissuesdevelop by nature in a development-dependent fashion.

It can be advantageously that the polypeptide of the present inventionis only active or has only an increased activity in the tissue which istransfected or penetrated by the pathogen mentioned herein. Especiallypreferred are constitutive promoters and leaf- and/or stem-specific,pathogen-inducible and epidermis-specific promoters, withpathogen-inducible and epidermis-specific promoters being mostpreferred. Also preferred is the natural promoter, which is e.g.comprised in the genomic fragment depicted in Seq. ID NO.: 5 and 6.

Furthermore, further promoters may be linked operatively to the nucleicacid sequence to be expressed, which promoters make possible theexpression in further plant tissues or in other organisms, such as, forexample, E. coli bacteria. Suitable plant promoters are, in principle,all of the above-described promoters.

The term “genetic control sequences” is to be understood in the broadsense and refers to also all those sequences which have an effect on thematerialization or the function of the expression cassette according tothe invention. For example, genetic control sequences modify thetranscription and translation in prokaryotic or eukaryotic organisms.Preferably, the expression cassettes according to the inventionencompass the promoter with specificity for the embryonic epidermisand/or the flower 5′-upstream of the nucleic acid sequence in questionto be expressed recombinantly, and 3′-downstream a terminator sequenceas additional genetic control sequence and, if appropriate, furthercustomary regulatory elements, in each case linked operatively to thenucleic acid sequence to be expressed recombinantly.

Genetic control sequences also encompass further promoters, promoterelements, or minimal promoters, all of which can modify theexpression-governing properties. Thus, for example, the tissue-specificexpression may additionally depend on certain stressors, owing togenetic control sequences. Such elements have been described, forexample, for water stress, abscisic acid (Lam E and Chua N H, J BiolChem 1991; 266(26): 17131-17135) and heat stress (Schoffl F et al.,Molecular & General Genetics 217(2-3):246-53, 1989).

Further advantageous control sequences are, for example, theGram-positive promoters amy and SPO2, and the yeast or fungal promotersADC1, MFa, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH.

In principle, all natural promoters with their regulatory sequences likethose mentioned above may be used for the method according to theinvention. In addition, synthetic promoters may also be usedadvantageously.

Genetic control sequences furthermore also encompass the 5′-untranslatedregions, introns or noncoding 3′-region of genes, such as, for example,the actin-1 intron, or the Adh1-S introns 1, 2, and 6 (generalreference: The Maize Handbook, Chapter 116, Freeling and Walbot, Eds.,Springer, N.Y. (1994)). It has been demonstrated that they may play asignificant role in the regulation of gene expression. Thus, it has beendemonstrated that 5′-untranslated sequences can enhance the transientexpression of heterologous genes. Examples of translation enhancerswhich may be mentioned are the tobacco mosaic virus 5′ leader sequence(Gallie et al. (1987) Nucl Acids Res 15:8693-8711) and the like.Furthermore, they may promote tissue specificity (Rouster J et al.(1998) Plant J 15:435-440).

The expression cassette may advantageously comprise one or more of whatare known as enhancer sequences, linked operatively to the promoter,which make possible an increased recombinant expression of the nucleicacid sequence. Additional advantageous sequences, such as furtherregulatory elements or terminators, may also be inserted at the 3′ endof the nucleic acid sequences to be expressed recombinantly. One or morecopies of the nucleic acid sequences to be expressed recombinantly maybe present in the gene construct.

In one embodiment the natural terminator sequence comprised in thegenomic fragment depicted in Seq ID No.: 5 and/or 6 is used.

Polyadenylation signals which are suitable as control sequences areplant polyadenylation signals, preferably those which essentiallycorrespond to T-DNA polyadenylation signals from Agrobacteriumtumefaciens, in particular gene 3′ of the T-DNA (octopin synthase) ofthe Ti plasmid pTiACHS (Gielen et al. (1984) EMBO J 3:835 et seq.) orfunctional equivalents thereof. Examples of terminator sequences whichare especially suitable are the OCS (octopin synthase) terminator andthe NOS (nopalin synthase) terminator.

Control sequences are furthermore to be understood as those which makepossible homologous recombination or insertion into the genome of a hostorganism or which permit removal from the genome. In the case ofhomologous recombination, for example the natural promoter of aparticular gene may be exchanged for a promoter with specificity for theembryonic epidermis and/or the flower. Methods such as the cre/loxtechnology permit a tissue-specific, if appropriate inducible, removalof the expression cassette from the genome of the host organism (Sauer B(1998) Methods. 14(4):381-92). In this method, specific flankingsequences (lox sequences), which later allow removal by means of crerecombinase, are attached to the target gene.

An expression cassette and the vectors derived from it may comprisefurther functional elements. The term functional element is to beunderstood in the broad sense and refers to all those elements whichhave an effect on the generation, amplification, or function of theexpression cassettes, vectors, or transgenic organisms according to theinvention. The following may be mentioned by way of example, but not bylimitation:

-   a) Selection markers which confer a resistance to a metabolism    inhibitor such as 2-deoxyglucose-6-phosphate (WO 98/45456),    antibiotics or biocides, preferably herbicides, such as, for    example, kanamycin, G 418, bleomycin or hygromycin, or else    phosphinothricin and the like. Especially preferred selection    markers are those which confer resistance to herbicides. Examples    which may be mentioned are: DNA sequences which encode    phosphinothricin acetyl transferases (PAT) and which inactivate    glutamine synthase inhibitors (bar and pat genes),    5-enolpyruvylshikimate-3-phosphate synthase genes (EPSP synthase    genes), which confer resistance to Glyphosater    (N-(phosphonomethyl)glycine), the gox gene, which encodes    Glyphosater-degrading enzymes (Glyphosate oxidoreductase), the deh    gene (encoding a dehalogenase which inactivates dalapon),    sulfonylurea- and imidazolinone-inactivating acetolactate synthases,    and bxn genes, which encode bromoxynil-degrading nitrilase enzymes,    the aasa gene, which confers resistance to the antibiotic    apectinomycin, the streptomycin phosphotransferase (SPT) gene, which    allows resistance to streptomycin, the neomycin phosphotransferase    (NPTII) gene, which confers resistance to kanamycin or geneticidin,    the hygromycin phosphotransferase (HPT) gene, which mediates    resistance to hygromycin, the acetolactate synthase gene (ALS),    which confers resistance to sulfonylurea herbicides (for example    mutated ALS variants with, for example, the S4 and/or Hra mutation).-   b) Reporter genes which encode readily quantifiable proteins and,    via their color or enzyme activity, make possible an assessment of    the transformation efficacy, the site of expression or the time of    expression. Very especially preferred in this context are genes    encoding reporter proteins (Schenborn E, Groskreutz D. Mol    Biotechnol. 1999; 13(1):29-44) such as the green fluorescent protein    (GFP) (Sheen et al. (1995) Plant Journal 8(5):777-784; Haseloff et    al. (1997) Proc Natl Acad Sci USA 94(6):2122-2127; Reichel et    al. (1996) Proc Natl Acad Sci USA 93(12):5888-5893; Tian et    al. (1997) Plant Cell Rep 16:267-271; WO 97/41228; Chui W L et    al. (1996) Curr Biol 6:325-330; Leffel S M et al. (1997)    Biotechniques. 23(5):912-8), chloramphenicol transferase, a    luciferase (Ow et al. (1986) Science 234:856-859; Millar et    al. (1992) Plant Mol Biol Rep 10:324-414), the aequorin gene    (Prasher et al. (1985) Biochem Biophys Res Commun 126(3):1259-1268),    β-galactosidase, R locus gene (encoding a protein which regulates    the production of anthocyanin pigments (red coloring) in plant    tissue and thus makes possible the direct analysis of the promoter    activity without addition of further auxiliary substances or    chromogenic substrates; Dellaporta et al., In: Chromosome Structure    and Function: Impact of New Concepts, 18th Stadler Genetics    Symposium, 11:263-282, 1988), with β-glucuronidase being very    especially preferred (Jefferson et al., EMBO J. 1987, 6, 3901-3907).-   c) Origins of replication, which ensure amplification of the    expression cassettes or vectors according to the invention in, for    example, E. coli. Examples which may be mentioned are ORI (origin of    DNA replication), the pBR322 ori or the P15A ori (Sambrook et al.:    Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor    Laboratory Press, Cold Spring Harbor, N.Y., 1989).-   d) Elements which are necessary for Agrobacterium-mediated plant    transformation, such as, for example, the right or left border of    the T-DNA or the vir region.

To select cells which have successfully undergone homologousrecombination, or else to select transformed cells, it is, as a rule,necessary additionally to introduce a selectable marker, which confersresistance to a biocide (for example herbicide), a metabolism inhibitorsuch as 2-deoxyglucose-6-phosphate (WO 98/45456) or an antibiotic to thecells which have successfully undergone recombination. The selectionmarker permits the selection of the transformed cells from untransformedones (McCormick et al. (1986) Plant Cell Reports 5:81-84).

The introduction of an expression cassette according to the inventioninto an organism or cells, tissues, organs, parts or seeds thereof(preferably into plants or plant cells, tissue, organs, parts or seeds)can be effected advantageously using vectors which comprise theexpression cassettes. The expression cassette can be introduced into thevector (for example a plasmid) via a suitable restriction cleavage site.The plasmid formed is first introduced into E. coli. Correctlytransformed E. coli are selected, grown, and the recombinant plasmid isobtained by the methods familiar to the skilled worker. Restrictionanalysis and sequencing may serve to verify the cloning step.

Further promoters for expression in specific plant parts are e.g. thenapin-gene promoter from rapeseed (U.S. Pat. No. 5,608,152), theUSP-promoter from Vicia faba (Baeumlein et al., Mol Gen Genet, 1991, 225(3):459-67), the oleosin-promoter from Arabidopsis (WO9845461), thephaseolin-promoter from Phaseolus vulgaris (U.S. Pat. No. 5,504,200),the Bce4-promoter from Brassica (WO9113980) or the legumin B4 promoter(LeB4; Baeumlein et al., 1992, Plant Journal, 2 (2):233-9) as well aspromoters conferring seed specific expression in monocot plants likemaize, barley, wheat, rye, rice etc. Suitable promoters to note are thelpt2 or lpt1-gene promoter from barley (WO9515389 and WO9523230) orthose described in WO9916890 (promoters from the barley hordein-gene,the rice glutelin gene, the rice oryzin gene, the rice prolamin gene,the wheat gliadin gene, wheat glutelin gene, the maize zein gene, theoat glutelin gene, the Sorghum kasirin-gene, the rye secalin gene).

Further, the polynucleotide of the invention can be cloned into theexpression vector in an antisense orientation. That is, the DNA moleculeis operatively linked to a regulatory sequence in a manner which allowsfor expression (by transcription of the DNA molecule) of an RNA moleculewhich is antisense to the mRNA encoded by the polynucleotide of thepresent invention. Regulatory sequences operatively linked to a nucleicacid cloned in the antisense orientation can be chosen which direct thecontinuous expression of the antisense RNA molecule in a variety of celltypes, for instance viral promoters and/or enhancers, or regulatorysequences can be chosen which direct constitutive, tissue specific orcell type specific expression of antisense RNA. The antisense expressionvector can be in the form of a recombinant plasmid, phagemid orattenuated virus in which antisense nucleic acid molecules are producedunder the control of a high efficiency regulatory region, the activityof which can be determined by the cell type into which the vector isintroduced. For a discussion of the regulation of gene expression usingantisense genes see Weintraub, H. et al., Antisense RNA as a moleculartool for genetic analysis, Reviews—Trends in Genetics, Vol. 1(1) 1986and Mol et al., 1990, FEBS Letters 268:427-430.

In one embodiment the present invention relates to a method of making arecombinant host cell comprising introducing the vector or thepolynucleotide of the present invention or said vector or saidpolynucleotide and a vector for expressing a further resistance proteininto a host cell.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection”, conjugation andtransduction are intended to refer to a variety of art-recognizedtechniques for introducing foreign nucleic acid (e.g., DNA) into a hostcell, including calcium phosphate or calcium chloride co-precipitation,DEAE-dextran-mediated transfection, lipofection, natural competence,chemical-mediated transfer, or electroporation. Suitable methods fortransforming or transfecting host cells including plant cells can befound in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd,ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1989) and other laboratory manuals such asMethods in Molecular Biology, 1995, Vol. 44, Agrobacterium protocols,ed: Gartland and Davey, Humana Press, Totowa, N.J.

For stable transfection of eukaryotic cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin, and methotrexate or in plants thatconfer resistance towards a herbicide such as glyphosate or glufosinate.Nucleic acid encoding a selectable marker can be introduced into a hostcell on the same vector as that encoding the polypeptide of the presentinvention or can be introduced on a separate vector. Cells stablytransfected with the introduced nucleic acid can be identified by, forexample, drug selection (e.g., cells that have incorporated theselectable marker gene will survive, while the other cells die).

Further host cells can be produced which contain selection systems whichallow for regulated expression of the introduced gene. For example,inclusion of the polynucleotide of the invention on a vector placing itunder control of the lac operon permits expression of the polynucleotideonly in the presence of IPTG. Such regulatory systems are well known inthe art.

Preferably, the introduced nucleic acid molecule is foreign to the hostcell.

By “foreign” it is meant that the nucleic acid molecule is eitherheterologous with, respect to the host cell, this means derived from acell or organism with a different genomic background, or is homologouswith respect to the host cell but located in a different genomicenvironment than the naturally occurring counterpart of said nucleicacid molecule. This means that, if the nucleic acid molecule ishomologous with respect to the host cell, it is not located in itsnatural location in the genome of said host cell, in particular it issurrounded by different genes. In this case the nucleic acid moleculemay be either under the control of its own promoter or under the controlof a heterologous promoter. The vector or nucleic acid moleculeaccording to the invention which is present in the host cell may eitherbe integrated into the genome of the host cell or it may be maintainedin some form extrachromosomally. In this respect, it is also to beunderstood that the nucleic acid molecule of the invention can be usedto restore or create a mutant gene via homologous recombination(Paszkowski (ed.), Homologous Recombination and Gene Silencing inPlants. Kluwer Academic Publishers (1994)).

Accordingly, in another embodiment the present invention relates to ahost cell genetically engineered with the polynucleotide of theinvention or the vector of the invention, or said vector or saidpolynucleotide and a vector or a polynucleotide for expressing a furtherresistance protein.

The terms “host cell” and “recombinant host cell” are usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but also to the progeny or potentialprogeny of such a cell. Because certain modifications may occur insucceeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term as usedherein.

For example, a polynucleotide of the present invention can be introducedin bacterial cells, insect cells, fungal cells or mammalian cells (suchas Chinese hamster ovary cells (CHO) or COS cells), algae, ciliates,plant cells or fungi. Suitable host cells are known to those skilled inthe art. Preferred are E. coli, baculovirus, Agrobacterium, or plantcells.

Further, the host cell can also be transformed such that further enzymesand proteins are (over)expressed which expression supports an increaseof resistance of a plant to pathogens. Preferably, a further resistancegene is also expressed, preferably one or more resistance genes,preferably the genes as mentioned herein, is/are also expressed. Mostpreferred is a coexpression of Rpi-blb2 and Rpi-blb.

Further preferred are cells of one of herein mentioned plants, inparticular, of one of the above-mentioned Solanaceae, most preferred arepotato, tomato, petunia, tree tomato, pear melon, or eggplant.

In another embodiment, the present invention relates to a process forthe production of the polypeptide of the present invention, inparticular of a protein having Rpi-blb2 activity comprising culturingthe host cell of the invention and recovering the polypeptide encoded bysaid polynucleotide and expressed by the host cell from the culture orthe cells.

The term “expression” means the production of a protein or nucleotidesequence in the cell. However, said term also includes expression of theprotein in a cell-free system. It includes transcription into an RNAproduct, post-transcriptional modification and/or translation to aprotein product or polypeptide from an DNA encoding that product, aswell as possible post-translational modifications.

Depending on the specific constructs and conditions used, the proteinmay be recovered from the cells, from the culture medium or from both.For the person skilled in the art it is well known that it is not onlypossible to express a native protein but also to express the protein asfusion polypeptides or to add signal sequences directing the protein tospecific compartments of the host cell, e.g., ensuring secretion of theprotein into the culture medium, etc. Furthermore, such a protein andfragments thereof can be chemically synthesized and/or modifiedaccording to standard methods described, for example herein below.

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) the polypeptideencoded by the polynucleotide of the invention, preferably a polypeptidhaving Rpi-blb2 activity. An alternate method can be applied in additionin plants by the direct transfer of DNA into developing flowers viaelectroporation or Agrobacterium mediated gene transfer. Accordingly,the invention further provides methods for producing Rpi-blb2 using thehost cells of the invention. In one embodiment, the method comprisesculturing the host cell of invention in a suitable medium such that thepolypeptid of the present invention is produced. Further, the methodcomprises isolating and/or recovering said polypeptid from the medium orthe host cell.

The polypeptide of the present invention is preferably produced byrecombinant DNA techniques. For example, a nucleic acid moleculeencoding the protein is cloned into an expression vector (as describedabove), the expression vector is introduced into a host cell (asdescribed above) and said polypeptide is expressed in the host cell.Said polypeptide can then be isolated from the cells by an appropriatepurification scheme using standard protein purification techniques.Alternative to recombinant expression, the polypeptide or peptide of thepresent invention can be synthesized chemically using standard peptidesynthesis techniques. Moreover, native Rpi-blb2 can be isolated fromcells (e.g., endothelial cells), for example using the antibody of thepresent invention as described below, in particular, an anti-Rpi-blb2antibody, which can be produced by standard techniques utilizing thepolypeptid of the present invention or fragment thereof, i.e., thepolypeptide of this invention.

In one embodiment, the present invention relates to a Rpi-blb2 proteinor a protein having Rpi-blb2 activity.

In one embodiment, the present invention relates to a polypeptide havingthe amino acid sequence encoded by a polynucleotide of the invention orobtainable by a process of the invention.

In one embodiment the polypeptide of the does not consist of thesequence depicted in Seq. ID NO.: 8 and/or 10 and/or does not consist ofthe sequence encoded by a nucleic acid molecule depicted in Seq. ID NO.:7 and/or 9.

In one embodiment, the polypeptide of the present invention does notconsist of the sequence of Mi1.1 or Mi.1.2 protein and/or of a proteinencoded by a nucleic acid molecule encoding a Mi1.1 or Mi1.2 protein.

Thus, in one embodiment, the polypeptide of the present invention maynot consist of the sequences shown in Rossi et al. 1998, PNAS USA95:9750-9754, Milligan et al., 1998. Plant Cell 10:1307-1319; and/or WO9806750.

The terms “protein” and “polypeptide” used in this application areinterchangeable. “Polypeptide” refers to a polymer of amino acids (aminoacid sequence) and does not refer to a specific length of the molecule.Thus peptides and oligopeptides are included within the definition ofpolypeptide. This term does also refer to or include post-translationalmodifications of the polypeptide, for example, glycosylations,acetylations, phosphorylations and the like. Included within thedefinition are, for example, polypeptides containing one or more analogsof an amino acid (including, for example, unnatural amino acids, etc.),polypeptides with substituted linkages, as well as other modificationsknown in the art, both naturally occurring and non-naturally occurring.

Preferably, the polypeptide is isolated. An “isolated” or “purified”protein or biologically active portion thereof is substantially free ofcellular material when produced by recombinant DNA techniques, orchemical precursors or other chemicals when chemically synthesized.

The language “substantially free of cellular material” includespreparations of the polypeptide of the invention in which the protein isseparated from cellular components of the cells in which it is naturallyor recombinantly produced. In one embodiment, the language“substantially free of cellular material” includes preparations havingless than about 30% (by dry weight) of “contaminating protein”, morepreferably less than about 20% of “contaminating protein”, still morepreferably less than about 10% of “contaminating protein”, and mostpreferably less than about 5% “contaminating protein”. The term“Contaminating protein” relates to polypeptides which are notpolypeptides of the present invention. When the polypeptide of thepresent invention or biologically active portion thereof isrecombinantly produced, it is also preferably substantially free ofculture medium, i.e., culture medium represents less than about 20%,more preferably less than about 10%, and most preferably less than about5% of the volume of the protein preparation. The language “substantiallyfree of chemical precursors or other chemicals” includes preparations inwhich subject of the present invention, e.g. the polypeptide of thepresent invention, is separated from chemical precursors or otherchemicals which are involved in the synthesis of the protein. Thelanguage “substantially free of chemical precursors or other chemicals”includes preparations having less than about 30% (by dry weight) ofchemical precursors or non-Rpi-blb2 chemicals, more preferably less thanabout 20% chemical precursors or non-Rpi-blb2 chemicals, still morepreferably less than about 10% chemical precursors or non-Rpi-blb2chemicals, and most preferably less than about 5% chemical precursors ornon-Rpi-blb2 chemicals. In preferred embodiments, isolated proteins orbiologically active portions thereof lack contaminating proteins fromthe same organism from which the polypeptide of the present invention isderived. Typically, such proteins are produced by recombinant DNAtechniques.

A polypeptide of the invention can participate in the polypeptide orportion thereof comprises preferably an amino acid sequence which issufficiently homologous to an amino acid sequence of SEQ ID No: 2 or 4such that the protein or portion thereof maintains the ability to conferthe resistance of the present invention. The portion of the protein ispreferably a biologically active portion as described herein.Preferably, the polypeptide of the invention has an amino acid sequenceidentical as shown in SEQ ID No: 2 or 4. Further, the polypeptide canhave an amino acid sequence which is encoded by a nucleotide sequencewhich hybridises, preferably hybridises under stringent conditions asdescribed above, to a nucleotide sequence of the polynucleotide of thepresent invention. Accordingly, the polypeptide has an amino acidsequence which is encoded by a nucleotide sequence that is at leastabout 70%, preferably at least about 75%, more preferably at least about80%, 90%, 95%, and even more preferably at least about 96%, 97%, 98%,99% or more homologous to one of the amino acid sequences of SEQ ID No:2 or 4. The preferred polypeptide of the present invention preferablypossess at least one of the Rpi-blb2 protein activities describedherein, e.g. its resistance or immunological activities. A preferredpolypeptide of the present invention includes an amino acid sequenceencoded by a nucleotide sequence which hybridises, preferably hybridisesunder stringent conditions, to a nucleotide sequence of SEQ ID No: 1 or3 or 5 or 6 or which is homologous thereto, as defined above.

Accordingly the polypeptide of the present invention can vary from SEQID No: 2, or 4 in amino acid sequence due to natural variation ormutagenesis, as described in detail herein. Accordingly, the polypeptidecomprise an amino acid sequence which is at least about 70%, preferablyat least about 75%, and more preferably at least about 80, 90, 95%, andmost preferably at least about 96%, 97%, 98%, 99% or more homologous toan entire amino acid sequence of SEQ ID No:1 or 3 or 5 or 6.

Biologically active portions of an polypeptide of the present inventioninclude peptides comprising amino acid sequences derived from the aminoacid sequence of a Rpi-blb2 protein, e.g., the amino acid sequence shownin SEQ ID No: 2 or 4 or the amino acid sequence of a protein homologousthereto, which include fewer amino acids than a full length Rpi-blb2protein or the full length protein which is homologous to a Rpi-blb2protein depicted herein, and exhibit at least one activity of Rpi-blb2protein. Typically, biologically (or immunological) active portions i.e.peptides, e.g., peptides which are, for example, 5, 10, 15, 20, 30, 35,36, 37, 38, 39, 40, 50, 100 or more amino acids in length comprise adomain or motif with at least one activity or epitope of a Rpi-blb2protein. Moreover, other biologically active portions, in which otherregions of the polypeptide are deleted, can be prepared by recombinanttechniques and evaluated for one or more of the activities describedherein.

Manipulation of the Rpi-blb2 polynucleotide of the invention may resultin the production of Rpi-blb2 having functional differences from thewild-type Rpi-blb2 protein. These proteins may be improved in efficiencyor activity, may be present in greater numbers in the cell than isusual, or may be decreased in efficiency or activity.

Any mutagenesis strategies for Rpi-blb2 to result in increased saidresistance or a resistance to another plant pathogen species or an otherstrain of a plant pathogen species aforementioned, of said compound arenot meant to be limiting; variations on these strategies will be readilyapparent to one skilled in the art. Using such strategies, andincorporating the mechanisms disclosed herein, the polynucleotide andpolypeptide of the invention may be utilized to generate plants or partsthereof; expressing wild type Rpi-blb2 or mutated Rpi-blb2polynucleotide and protein molecules such that the yield, production,and/or efficiency of production of a desired compound is improved. Thisdesired compound may be any natural product of plants, which includesthe final products of biosynthesis pathways and intermediates ofnaturally-occurring metabolic pathways, as well as molecules which donot naturally occur in the metabolism of said cells, but which areproduced by a said cells of the invention.

The invention also provides chimeric or fusion proteins.

As used herein, a “chimeric protein” or “fusion protein” comprises anpolypeptide operatively linked to a non-Rpi-blb2 polypeptide.

An “Rpi-blb2 polypeptide” refers to a polypeptide having an amino acidsequence corresponding to polypeptide having a Rpi-blb2 activity,whereas a “non-Rpi-blb2 polypeptide” refers to a polypeptide having anamino acid sequence corresponding to a protein which is notsubstantially homologous to the Rpi-blb2, e.g., a protein which does notconfer the resistance described herein, in particular does not conferresistance to P. infestans and which is derived from the same or adifferent organism. Within the fusion protein, the term “operativelylinked” is intended to indicate that the Rpi-blb2 polypeptide and thenon-Rpi-blb2 polypeptide are fused to each other so that both sequencesfulfil the proposed function addicted to the sequence used. Thenon-Rpi-blb2 polypeptide can be fused to the N-terminus or C-terminus ofthe Rpi-blb2 polypeptide. For example, in one embodiment the fusionprotein is a GST-LMRP fusion protein in which the Rpi-blb2 sequences arefused to the C-terminus of the GST sequences. Such fusion proteins canfacilitate the purification of recombinant Rpi-blb2. In anotherembodiment, the fusion protein is a Rpi-blb2 containing a heterologoussignal sequence at its N-terminus. In certain host cells (e.g.,mammalian host cells), expression and/or secretion of a Rpi-blb2 can beincreased through use of a heterologous signal sequence.

Preferably, a Rpi-blb2 chimeric or fusion protein of the invention isproduced by standard recombinant DNA techniques. For example, DNAfragments coding for the different polypeptide sequences are ligatedtogether in-frame in accordance with conventional techniques, forexample by employing blunt-ended or stagger-ended termini for ligation,restriction enzyme digestion to provide for appropriate termini,filling-in of cohesive ends as appropriate, alkaline phosphatasetreatment to avoid undesirable joining, and enzymatic ligation. Thefusion gene can be synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers which give rise tocomplementary overhangs between two consecutive gene fragments which cansubsequently be annealed and reamplified to generate a chimeric genesequence (see, for example, Current Protocols in Molecular Biology, eds.Ausubel et al. John Wiley & Sons: 1992). Moreover, many expressionvectors are commercially available that already encode a fusion moiety(e.g., a GST polypeptide). The polynucleotide of the invention can becloned into such an expression vector such that the fusion moiety islinked in-frame to the encoded protein.

Furthermore, folding simulations and computer redesign of structuralmotifs of the protein of the invention can be performed usingappropriate computer programs (Olszewski, Proteins 25 (1996), 286-299;Hoffman, Comput. Appl. Biosci. 11 (1995), 675-679). Computer modellingof protein folding can be used for the conformational and energeticanalysis of detailed peptide and protein models (Monge, J. Mol. Biol.247 (1995), 995-1012; Renouf, Adv. Exp. Med. Biol. 376 (1995), 37-45).In particular, the appropriate programs can be used for theidentification of interactive sites of mitogenic cyplin and itsreceptor, its ligand or other interacting proteins by computer assistantsearches for complementary peptide sequences (Fassina, Immunomethods(1994), 114-120. Further appropriate computer systems for the design ofprotein and peptides are described in the prior art, for example inBerry, Biochem. Soc. Trans. 22 (1994), 1033-1036; Wodak, Ann. N.Y. Acad.Sci. 501 (1987), 1-13; Pabo, Biochemistry 25 (1986), 5987-5991. Theresults obtained from the above-described computer analysis can be usedfor, e.g., the preparation of peptidomimetics of the protein of theinvention or fragments thereof. Such pseudopeptide analogues of the,natural amino acid sequence of the protein may very efficiently mimicthe parent protein (Benkirane, J. Biol. Chem. 271 (1996), 33218-33224).For example, incorporation of easily available achiral Q-amino acidresidues into a protein of the invention or a fragment thereof resultsin the substitution of amide bonds by polymethylene units of analiphatic chain, thereby providing a convenient strategy forconstructing a peptidomimetic (Banerjee, Biopolymers 39 (1996),769-777).

Superactive peptidomimetic analogues of small peptide hormones in othersystems are described in the prior art (Zhang, Biochem. Biophys. Res.Commun. 224 (1996), 327-331). Appropriate peptidomimetics of the proteinof the present invention can also be identified by the synthesis ofpeptidomimetic combinatorial libraries through successive amidealkylation and testing the resulting compounds, e.g., for their bindingand immunological properties. Methods for the generation and use ofpeptidomimetic combinatorial libraries are described in the prior art,for example in Ostresh, Methods in Enzymology 267 (1996), 220-234 andDorner, Bioorg. Med. Chem. 4 (1996), 709-715.

Furthermore, a three-dimensional and/or crystallographic structure ofthe protein of the invention can be used for the design ofpeptidomimetic inhibitors of the biological activity of the protein ofthe invention (Rose, Biochemistry 35 (1996), 12933-12944; Rutenber,Bioorg. Med. Chem. 4 (1996), 1545-1558).

In a further embodiment, the present invention relates to an antibodythat binds specifically to the polypeptide of the present invention orparts, i.e. specific fragments or epitopes of such a protein.

The antibodies of the invention can be used to identify and isolateRpi-blb2 and genes in any organism, preferably plants, prepared inplants described herein. These antibodies can be monoclonal antibodies,polyclonal antibodies or synthetic antibodies as well as fragments ofantibodies, such as Fab, Fv or scFv fragments etc. Monoclonal antibodiescan be prepared, for example, by the techniques as originally describedin Köhler and Milstein, Nature 256 (1975), 495, and Galfr6, Meth.Enzymol. 73 (1981), 3, which comprise the fusion of mouse myeloma cellsto spleen cells derived from immunized mammals.

Furthermore, antibodies or fragments thereof to the aforementionedpeptides can be obtained by using methods which are described, e.g., inHarlow and Lane “Antibodies, A Laboratory Manual”, CSH Press, ColdSpring Harbor, 1988. These antibodies can be used, for example, for theimmunoprecipitation and immunolocalization of proteins according to theinvention as well as for the monitoring of the synthesis of suchproteins, for example, in recombinant organisms, and for theidentification of compounds interacting with the protein according tothe invention. For example, surface plasmon resonance as employed in theBIAcore system can be used to increase the efficiency of phageantibodies selections, yielding a high increment of affinity from asingle library of phage antibodies which bind to an epitope of theprotein of the invention (Schier, Human Antibodies Hybridomas 7 (1996),97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13). In many cases,the binding phenomena of antibodies to antigens is equivalent to otherligand/anti-ligand binding.

In one embodiment, the present invention relates to an antisense nucleicacid molecule comprising the complementary sequence of the polypeptideof the present invention.

Methods to modify the expression levels and/or the activity are known topersons skilled in the art and include for instance overexpression,co-suppression, the use of ribozymes, sense and anti-sense strategies,gene silencing approaches. “Sense strand” refers to the strand of adouble-stranded DNA molecule that is homologous to a mRNA transcriptthereof. The “anti-sense strand” contains an inverted sequence which iscomplementary to that of the “sense-strand”.

An “antisense” nucleic acid molecule comprises a nucleotide sequencewhich is complementary to a “sense” nucleic acid molecule encoding aprotein, e.g., complementary to the coding strand of a double-strandedcDNA molecule or complementary to an mRNA sequence. Accordingly, anantisense nucleic acid molecule can hydrogen bond to a sense nucleicacid molecule. The antisense nucleic acid molecule can be complementaryto an entire Rpi-blb2 coding strand, or to only a portion thereof.Accordingly, an antisense nucleic acid molecule can be antisense to a“coding region” of the coding strand of a nucleotide sequence of apolynucleotide of the present invention. The term “coding region” refersto the region of the nucleotide sequence comprising codons which aretranslated into amino acid residues. Further, the antisense nucleic acidmolecule is antisense to a “noncoding region” of the coding strand of anucleotide sequence encoding Rpi-blb2. The term “noncoding region”refers to 5′ and 3′ sequences which flank the coding region that are nottranslated into a polypeptide, i.e., also referred to as 5′ and 3′untranslated regions (5′-UTR or 3′-UTR).

Given the coding strand sequences encoding Rpi-blb2 disclosed herein,antisense nucleic acid molecules of the invention can be designedaccording to the rules of Watson and Crick base pairing. The antisensenucleic acid molecule can be complementary to the entire coding regionof Rpi-blb2 mRNA, but can also be an oligonucleotide which is antisenseto only a portion of the coding or noncoding region of Rpi-blb2 mRNA.For example, the antisense oligonucleotide can be complementary to theregion surrounding the translation start site of Rpi-blb2 mRNA. Anantisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25,30, 35, 40, 45, or 50 nucleotides in length. An antisense nucleic acidmolecule of the invention can be constructed using chemical synthesisand enzymatic ligation reactions using procedures known in the art. Forexample, an antisense nucleic acid molecule (e.g., an antisenseoligonucleotide) can be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed between the antisense and sense nucleicacids, e.g., phosphorothioate derivatives and acridine substitutednucleotides can be used. Examples of modified nucleotides which can beused to generate the antisense nucleic acid include 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,4-acetylcytosine, 5-(carboxyhydroxylmethyl)racil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which apolynucleotide has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted polynucleotide will be of an antisenseorientation to a target polynucleotide of interest, described further inthe following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a cell or generated in situ such that they hybridisewith or bind to cellular mRNA and/or genomic DNA encoding a Rpi-blb2 tothereby inhibit expression of the protein, e.g., by inhibitingtranscription and/or translation. The hybridisation can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule which bindsto DNA duplexes, through specific interactions in the major groove ofthe double helix. The antisense molecule can be modified such that itspecifically binds to a receptor or an antigen expressed on a selectedcell surface, e.g., by linking the antisense nucleic acid molecule to apeptide or an antibody which binds to a cell surface receptor orantigen. The antisense nucleic acid molecule can also be delivered tocells using the vectors described herein. To achieve sufficientintracellular concentrations of the antisense molecules, vectorconstructs in which the antisense nucleic acid molecule is placed underthe control of a strong prokaryotic, viral, or eukaryotic includingplant promoters are preferred.

In a further embodiment, the antisense nucleic acid molecule of theinvention can be an anomeric nucleic acid molecule. An α-anomericnucleic acid molecule forms specific double-stranded hybrids withcomplementary RNA in which, contrary to the usual units, the strands runparallel to each other (Gaultier et al. (1987) Nucleic Acids. Res.15:6625-6641). The antisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBSLett. 215:327-330).

Further the antisense nucleic acid molecule of the invention can be aribozyme. Ribozymes are catalytic RNA molecules with ribonucleaseactivity which are capable of cleaving a single-stranded nucleic acid,such as an mRNA, to which they have a complementary region. Thus,ribozymes (e.g., hammerhead ribozymes (described in Haselhoff andGerlach (1988) Nature 334:585-591)) can be used to catalytically cleaveRpi-blb2 mRNA transcripts to thereby inhibit translation of mRNA. Aribozyme having specificity for a Rpi-blb2-encoding nucleic acidmolecule can be designed based upon the nucleotide sequence of aRpi-blb2 cDNA disclosed herein or on the basis of a heterologoussequence to be isolated according to methods taught in this invention.For example, a derivative of a Tetrahymena L-19 IVS RNA can beconstructed in which the nucleotide sequence of the active site iscomplementary to the nucleotide sequence to be cleaved in an encodingmRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071 and Cech et al.U.S. Pat. No. 5,116,742. Alternatively, Rpi-blb2 mRNA can be used toselect a catalytic RNA having a specific ribonuclease activity from apool of RNA molecules. See, e.g., Bartel, D. and Szostak, J. W. (1993)Science 261:1411-1418.

The antisense molecule of the present invention comprises also apolynucleotide comprising a nucleotide sequences complementary to theregulatory region of a Rpi-blb2 nucleotide sequence, e.g., its promoterand/or enhancers, e.g. to form triple helical structures that preventtranscription of the gene in target cells. See generally, Helene, C.(1991) Anticancer Drug Des. 6(6):569-84; Helene, C. et al. (1992) Ann.N.Y. Acad. Sci. 660:27-36; and Maher, L. J. (1992) Bioassays14(12):807-15.

In addition, in one embodiment, the present invention relates to amethod for the production of transgenic plants, plant cells or planttissue comprising the introduction of the polynucleotide or the vectorof the present invention into the genome of said plant, plant tissue orplant cell. In a preferred embodiment, said vector or saidpolynucleotide and a vector or a polynucleotide for the expression of afurther resistance gene, in particular for Rpi-blb, is also introducedinto the genome of said plant, plant tissue or plant cell, before, afteror together.

For the expression of the nucleic acid molecules according to theinvention in sense or antisense orientation in plant cells, themolecules are placed under the control of regulatory elements whichensure the expression in plant cells. These regulatory elements may beheterologous or homologous with respect to the nucleic acid molecule tobe expressed as well with respect to the plant species to be transformedand are described above in detail.

In general, such regulatory elements comprise a promoter active in plantcells. To obtain expression in all tissues of a transgenic plant, e.g.constitutive promoters are used, such as the 35 S promoter of CaMV(Odell, Nature 313 (1985), 810-812) or promoters of the polyubiquitingenes of maize (Christensen, Plant Mol. Biol. 18 (1982), 675-689). Inorder to achieve expression in specific tissues of a transgenic plant itis possible to use tissue specific promoters (see, e.g., Stockhaus, EMBOJ. 8 (1989), 2245-2251). Known are also promoters which are specificallyactive in tubers of potatoes or in seeds of different plants species,such as maize, Vicia, wheat, barley etc. Inducible promoters may be usedin order to be able to exactly control expression. Inducible promoterscomprise also promoters, which are induced by infections of plants.Further embodiments are described above.

In one embodiment, the present invention relates to a method forproducing a plant or a part thereof resistant to a pathogen of thephylum Oomycetes comprising the steps: expressing in the plant or a partthereof the polypeptide of the present invention and a furtherresistance protein.

Accordingly in one further embodiment, the present invention relates totransgenic plant or plant tissue of the invention or produced accordingto the method of the invention, which upon the presence of thepolynucleotide or the vector is resistant to said pathogens.

The generation of a transformed organism (or of a transformed cell ortissue) requires introducing the DNA, RNA, or protein in question intothe relevant host cell. A multiplicity of methods are available for thisprocedure, which is termed transformation (or transduction ortransfection) (Keown et al. (1990) Methods in Enzymology 185:527-537).For example, the DNA or RNA can be introduced directly by microinjectionor by bombardment with DNA-coated microparticles. Also, the cell can bepermeabilized chemically, for example using polyethylene glycol, so thatDNA can enter the cell by diffusion. The DNA can also be introduced byprotoplast fusion with other DNA-containing units such as minicells,cells, lysosomes, or liposomes. Another suitable method of introducingDNA is electroporation, where the cells are permeabilized reversibly byan electrical pulse. Suitable methods have been described (for exampleby Bilang et al. (1991) Gene 100:247-250; Scheid et al. (1991) Mol GenGenet 228:104-112; Guerche et al. (1987) Plant Science 52:111-116;Neuhause et al. (1987) Theor Appl Genet 75:30-36; Klein et al. (1987)Nature 327:70-73; Howell et al. (1980) Science 208:1265; Horsch et al.(1985) Science 227:1229-1231; DeBlock et al. (1989) Plant Physiology91:694-701; Methods for Plant Molecular Biology (Weissbach andWeissbach, eds.) Academic Press Inc. (1988); and Methods in PlantMolecular Biology (Schuler and Zielinski, eds.) Academic Press Inc.(1989)).

In plants, the above-described methods of transforming and regeneratingplants from plant tissues or plant cells are exploited for transient orstable transformation. Suitable methods are especially protoplasttransformation by polyethylene-glycol-induced DNA uptake, the ballisticmethod with the gene gun, what is known as the particle bombardmentmethod, electroporation, incubation of dry embryos in DNA-containingsolution, and microinjection.

In addition to these “direct” transformation techniques, transformationcan also be effected by bacterial infection by means of Agrobacteriumtumefaciens or Agrobacterium rhizogenes. The Agrobacterium-mediatedtransformation is best suited to dicotyledonous plant cells. The methodsare described, for example, by Horsch R B et al. (1985) Science 225:1229f.

When agrobacteria are used, the expression cassette must be integratedinto specific plasmids, either into a shuttle or intermediate vector, orinto a binary vector. If a Ti or Ri plasmid is to be used for thetransformation, at least the right border, but in most cases the rightand left border, of the Ti or Ri plasmid T-DNA is linked to theexpression cassette to be introduced in the form of a flanking region.

Binary vectors are preferably used. Binary vectors are capable ofreplication both in E. coli and in Agrobacterium. As a rule, theycomprise a selection marker gene and a linker or polylinker flanked bythe right and left T-DNA border sequence. They can be transferreddirectly into Agrobacterium (Holsters et al. (1978) Mol Gen Genet163:181-187). The selection marker gene permits the selection oftransformed agrobacteria and is, for example, the nptII gene, whichconfers resistance to kanamycin. The Agrobacterium which acts as hostorganism in this case should already contain a plasmid with the virregion. The latter is required for transferring the T-DNA to the plantcell. An Agrobacterium transformed in this way can be used fortransforming plant cells. The use of T-DNA for transforming plant cellshas been studied and described intensively (EP 120 516; Hoekema, In: TheBinary Plant Vector System, Offsetdrukkerij Kanters B. V., Alblasserdam,Chapter V; An et al. (1985) EMBO J 4:277-287). Various binary vectorsare known, some of which are commercially available such as, forexample, pBI101.2 or pBIN19 (Clontech Laboratories, Inc. USA).

Further promoters which are suitable for expression in plants have beendescribed (Rogers et al. (1987) Meth in Enzymol 153:253-277; Schardl etal. (1987) Gene 61:1-11; Berger et al. (1989) Proc Natl Acad Sci USA86:8402-8406).

Direct transformation techniques are suitable for any organism and celltype.

The plasmid used need not meet any particular requirements in the caseof the injection or electroporation of DNA or RNA into plant cells.Simple plasmids such as those of the pUC series can be used. If completeplants are to be regenerated from the transformed cells, it is necessaryfor an additional selectable marker gene to be located on the plasmid.

Stably transformed cells, i.e. those which contain the introduced DNAintegrated into the DNA of the host cell, can be selected fromuntransformed cells when a selectable marker is part of the DNAintroduced. Examples of genes which can act as markers are all thosewhich are capable of conferring resistance to antibiotics or herbicides(such as kanamycin, G 418, bleomycin, hygromycin or phosphinothricin)(see above). Transformed cells which express such marker genes arecapable of surviving in the presence of concentrations of acorresponding antibiotic or herbicide which kill an untransformed wildtype. Examples are mentioned above and preferably comprise the bar gene,which confers resistance to the herbicide phosphinothricin (Rathore K Set al. (1993) Plant Mol Biol 21(5):871-884), the nptil gene, whichconfers resistance to kanamycin, the hpt gene, which confers resistanceto hygromycin, or the EPSP gene, which confers resistance to theherbicide Glyphosate. The selection marker permits the selection oftransformed cells from untransformed cells (McCormick et al. (1986)Plant Cell Reports 5:81-84). The resulting plants can be bred andhybridised in the customary fashion. Two or more generations should begrown in order to ensure that the genomic integration is stable andhereditary.

The abovementioned methods are described, for example, in Jenes B et al.(1993) Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1,Engineering and Utilization, edited by S D Kung and R Wu, AcademicPress, pp. 128-143 and in Potrykus (1991) Annu Rev Plant Physiol PlantMolec Biol 42:205-225). The construct to be expressed is preferablycloned into a vector which is suitable for the transformation ofAgrobacterium tumefaciens, for example pBin19 (Bevan et al. (1984) NuclAcids Res 12:8711f).

As soon as a transformed plant cell has been generated, a complete plantcan be obtained using methods known to the skilled worker. For example,callus cultures are used as starting material. The development of shootand root can be induced in this as yet undifferentiated cell biomass ina known fashion. The shoots obtained can be planted out and bred.

The skilled worker is familiar with such methods of regenerating intactplants from plant cells and plant parts. Methods to do so are described,for example, by Fennell et al. (1992) Plant Cell Rep. 11: 567-570;Stoeger et al (1995) Plant Cell Rep. 14:273-278; Jahne et al. (1994)Theor Appl Genet 89:525-533.

The method according to the invention can advantageously be combinedwith further methods which bring about pathogen resistance (for exampleto insects, fungi, bacteria, nematodes and the like), stress resistanceor another improvement of the plant properties. Examples are mentioned,inter alia, by Dunwell J M, Transgenic approaches to crop improvement, JExp Bot. 2000; 51 Spec No; pages 487-96.

Suitable strains of Agrobacterium tumefaciens and vectors as well astransformation of Agrobacteria and appropriate growth and selectionmedia are well known to those skilled in the art and are described inthe prior art (GV31 01 (pMK90RK), Koncz, Mol. Gen. Genet. 204 (1986),383-396; C58C1 (pGV 3850kan), Deblaere, Nucl. Acid Res. 13 (1985), 4777;Bevan, Nucleic. Acid Res. 12(1984), 8711; Koncz, Proc. Natl. Acad. Sci.USA 86 (1989), 8467-8471; Koncz, Plant Mol. Biol. 20 (1992), 963-976;Koncz, Specialized vectors for gene tagging and expression studies. In:Plant Molecular Biology Manual Vol 2, Gelvin and Schilperoort (Eds.),Dordrecht, The Netherlands: Kluwer Academic Publ. (1994), 1-22; EP-A-120516; Hoekema: The Binary Plant Vector System, Offsetdrukkerij Kanters B.V., Alblasserdam (1985), Chapter V, Fraley, Crit. Rev. Plant. Sci., 4,1-46; An, EMBO J. 4 (1985), 277-287).

Although the use of Agrobacterium tumefaciens is preferred in the methodof the invention, other Agrobacterium strains, such as Agrobacteriumrhizogenes, may be used, for example if a phenotype conferred by saidstrain is desired.

The transformation of most dicotyledonous plants is possible with themethods described above. But also for the transformation ofmonocotyledonous plants several successful transformation techniqueshave been developed. These include the transformation using biolisticmethods as, e.g., described above as well as protoplast transformation,electroporation of partially permeabilized cells, introduction of DNAusing glass fibers, etc.

The term “transformation” as used herein, refers to the transfer of anexogenous polynucleotide into a host cell, irrespective of the methodused for the transfer. The polynucleotide may be transiently or stablyintroduced into the host cell and may be maintained non-integrated, forexample, as a plasmid or as chimeric links, or alternatively, may beintegrated into the host genome. The resulting transformed plant cellcan then be used to regenerate a transformed plant in a manner known bya skilled person.

Accordingly, in one embodiment, the present invention relates to a plantcell comprising the polynucleotide the vector of the present inventionor obtainable by the method of the present invention. Preferably, thecell comprises a further resistance conferring polynucleotide or vector,more preferred is a Rpi-blb encoding vector or polynucleotide.

Thus, the present invention relates also to transgenic plant cells whichcontain (preferably stably integrated into the genome) a polynucleotideaccording to the invention linked to regulatory elements which allowexpression of the polynucleotide in plant cells and wherein thepolynucleotide is foreign to the transgenic plant cell. For the meaningof foreign; see supra.

Thus, the present invention also relates to transgenic plants and planttissue comprising transgenic plant cells according to the invention. Dueto the (over)expression of a polypeptide of the invention, said plant orplant tissues are resistance to plant pathogens, in particular toOomycetes. Preferably the plants are also resistance to other pathogen,e.g. to sucking plant pathogens. Further pathogens are described herein.Preferred is that said plants or plant tissue is resistant toPhytophthora species, most preferred to P. infestans.

For example, to obtain transgenic plants expressing the Rpi-blb2 gene,its coding region can be cloned, e.g., into the pBinAR vector (Höfgenund Willmitzer, Plant-Science, 66, 1990, 221-230). For example,following a polymerase chain reaction (PCR) technology the coding regionof Rpi-blb2 can be amplified using Primers as shown in the examples andfigures, e.g., in Table 3b in particular ARF1F and ARF1R. The obtainedPCR fragment can be purified and subsequently the fragment can be clonedinto a vector. The resulted vector can be transferred into Agrobacteriumtumefaciens. This strain can be used to transform and transgenic plantscan then be selected. In another embodiment, the present inventionrelates to a transgenic plant or plant tissue comprising the plant cellof the present invention.

“Transgenic”, for example regarding a nucleic acid sequence, anexpression cassette or a vector comprising said nucleic acid sequence oran organism transformed with said nucleic acid sequence, expressioncassette or vector, refers to all those constructs originating byrecombinant methods in which either

-   a) the Rpi-blb2 nucleic acid sequence, or-   b) a genetic control sequence linked operably to the Rpi-blb2    nucleic acid sequence, for example a promoter, or-   c) (a) and (b)    are not located in their natural genetic environment or have been    modified by recombinant methods, an example of a modification being    a substitution, addition, deletion, inversion or insertion of one or    more nucleotide residues. Natural genetic environment refers to the    natural chromosomal locus in the organism of origin, or to the    presence in a genomic library. In the case of a genomic library, the    natural genetic environment of the nucleic acid sequence is    preferably retained, at least in part. The environment flanks the    nucleic acid sequence at least at one side and has a sequence of at    least 50 bp, preferably at least 500 bp, especially preferably at    least 1000 bp, very especially preferably at least 5000 bp, in    length. A naturally occurring expression cassette—for example the    naturally occurring combination of the Rpi-blb2 promoter with the    corresponding Rpi-blb2 gene—becomes a transgenic expression cassette    when it is modified by non-natural, synthetic “artificial” methods    such as, for example, mutagenization. Such methods have been    described (U.S. Pat. No. 5,565,350; WO 00/15815; also see above).

Further, the plant cell, plant tissue or plant can also be transformedsuch that further enzymes and proteins are (over)expressed whichexpression supports an increase of the plant's or the plant tissue'sresistance, for example Rpi-blb (synonyms Rpi-blb1, RB or Sbu1), R1,Rpi-mcd, R-ber (synonym R12), Rpi1, Rpi-blb3, Rpi-ABPT1, R2, R3a or R3b,R4, R5, R6, R7, R8, R9, R10, R11, Ph-1, Ph-2 and/or Ph-3-proteins.Preferred is the coexpression of Rpi-blb and Rpi-blb2.

The present invention also relates to cultured plant tissues comprisingtransgenic plant cells as described above which show expression of aprotein according to the invention.

Host or starting organisms which are preferred as transgenic organismsare mainly plants in accordance with the above definition. Includedwithin the scope of the invention are all genera and species of higherand lower plants of the Plant Kingdom. Furthermore included are themature plants, seed, shoots and seedlings, and parts, propagationmaterial and cultures derived there from, for example cell cultureswhich have an increased Rpi-blb2 activity. Mature plants refers toplants at any developmental stage beyond that of the seedling. The termseedling refers to a young immature plant in an early developmentalstage.

Any transformed plant obtained according to the invention can be used ina conventional breeding scheme or in in vitro plant propagation toproduce more transformed plants with the same characteristics and/or canbe used to introduce the same characteristic in other varieties of thesame or related species. Such plants are also part of the invention.Seeds obtained from the transformed plants genetically also contain thesame characteristic and are part of the invention. As mentioned before,the present invention is in principle applicable to any plant and cropthat can be transformed with any of the transformation method known tothose skilled in the art.

In general, the plants which can be modified according to the inventionand which either show overexpression of a protein according to theinvention or a reduction of the synthesis of such a protein can bederived from any desired plant species. They can be monocotyledonousplants or dicotyledonous plants, preferably they belong to plant speciesof interest in agriculture, wood culture or horticulture interest, suchas crop plants (e.g. maize, rice, barley, wheat, rye, oats etc.),potatoes, oil producing plants (e.g. oilseed rape, sunflower, pea nut,soy bean, etc.), cotton, sugar beet, sugar cane, leguminous plants (e.g.beans, peas etc.), wood producing plants, preferably trees, etc.However, plants which can be infected by Phytophthora species arepreferred.

Accordingly, in one embodiment the plant, plant cell or plant tissue ofthe invention or produced according to the method of the invention isselected from the group consisting of Menyanthaceae, Solanaceae,Sclerophylacaceae, Duckeodendraceae, Goetzeaceae, Convolvulaceae,Cuscutaceae, Polemoniaceae, and Hydrophyllaceae according to the SystemaNaturae 2000, Brands, S. J., Amsterdam or has its origin thereof.Preferably said plant, plant cell or plant tissue of the invention orproduced according to the method of the invention is a Solanaceae,preferably selected from the group of Atropa, Browallia, Brunfelsia,Capsicum, Cestrum, Cyphomandra, Datura, Fabiana, Franciscea, Hyoscyamus,Lycium, Mandragora, Nicandra, Nicotiana, Petunia, Physalis, Schizanthusand Solanum according to the Systema Naturae 2000, Brands, S. J.,Amsterdam or has its origin thereof.

More preferred, the plant, plant cell or plant tissue of the inventionor produced according to the method of the present invention is a S.bulbocastanum, S. tuberosum (potato), S. lycopersicum (tomato), petunia,S. betaceum (tree tomato), S. muricatum (pear melon) or S. melongena(eggplant). Even more preferred, the plant, plant tissue or plant cellis a S. tuberosum or S. lycopersicum. Most preferred is S. tuberosum. Inother systems, the classification will be similar. The person skilled inthe art knows the differences, e.g. more common, tomato is namedsystematically Lycopersicon lycopersicum (L.) Karsten ex Farwell.

In yet another aspect, the invention also relates to harvestable partsand to propagation material of the transgenic plants according to theinvention which either contain transgenic plant cells expressing anucleic acid molecule and/or the polypeptide according to the inventionor which contains cells which show an increased level of the polypeptideof the invention.

Harvestable parts can be in principle any useful parts of a plant, forexample, flowers, pollen, seedlings, tubers, leaves, stems, fruit,seeds, roots etc. Propagation material includes, for example, seeds,fruits, cuttings, seedlings, tubers, rootstocks etc. Preferred arepotatoes, tomatoes, eggfruits or pear melons as harvestable orpropagation material. In case, the plant of the invention is petunia,the present invention relates in one embodiment to the flowers ofpetunia as harvestable part.

The invention furthermore relates to the use of the transgenic organismsaccording to the invention and of the cells, cell cultures, parts—suchas, for example, roots, leaves and the like in the case of transgenicplant organisms—derived from them, and to transgenic propagationmaterial such as seeds or fruits, for the production of foodstuffs orfeeding stuffs, pharmaceuticals or fine chemicals. In particular,potatoes can serve for the production of fine chemicals.

Accordingly in another embodiment, the present invention relates to theuse of the polynucleotide, the plant, plant cell or plant tissue, thevector, or the polypeptide of the present invention for making fattyacids, carotenoids, isoprenoids, vitamins, lipids, wax esters,(poly)saccharides and/or polyhydroxyalkanoates, and/or its metabolismproducts, in particular, steroid hormones, cholesterol, prostaglandin,triacylglycerols, bile acids and/or ketone bodies producing cells,tissues and/or plants. There are a number of mechanisms by which theyield, production, and/or efficiency of production of fatty acids,carotenoids, isoprenoids, vitamins, wax esters, lipids,(poly)saccharides and/or polyhydroxyalkanoates, and/or its metabolismproducts, in particular, steroid hormones, cholesterol,triacylglycerols, prostaglandin, bile acids and/or ketone bodies orfurther of above defined fine chemicals incorporating such an alteredprotein can be affected. In the case of plants, by e.g. increasing theexpression of acetyl-CoA which is the basis for many products, e.g.,fatty acids, carotenoids, isoprenoids, vitamins, lipids,(poly)saccharides, wax esters, and/or polyhydroxyalkanoates, and/or itsmetabolism products, in particular, prostaglandin, steroid hormones,cholesterol, triacylglycerols, bile acids and/or ketone bodies in acell, it may be possible to increase the amount of the produced saidcompounds thus permitting greater ease of harvesting and purification orin case of plants more efficient partitioning. Further, one or more ofsaid metabolism products, increased amounts of the cofactors, precursormolecules, and intermediate compounds for the appropriate biosyntheticpathways maybe required. Therefore, by increasing the number and/oractivity of transporter proteins involved in the import of nutrients,such as carbon sources (i.e., sugars), nitrogen sources (i.e., aminoacids, ammonium salts), phosphate, and sulphur, it may be possible toimprove the production of acetyl CoA and its metabolism products asmentioned above, due to the removal of any nutrient supply limitationson the biosynthetic process. In particular, it may be possible toincrease the yield, production, and/or efficiency of production of saidcompounds, e.g. fatty acids, carotenoids, isoprenoids, vitamins, wasesters, lipids, (poly)saccharides, and/or polyhydroxyalkanoates, and/orits metabolism products, in particular, steroid hormones, cholesterol,prostaglandin, triacylglycerols, bile acids and/or ketone bodiesmolecules etc. in plants.

Furthermore preferred is a method for the recombinant production ofpharmaceuticals or fine chemicals in host organisms, wherein a hostorganism is transformed with one of the above-described expressioncassettes and this expression cassette comprises one or more structuralgenes which encode the desired fine chemical or catalyse thebiosynthesis of the desired fine chemical, the transformed host organismis cultured, and the desired fine chemical is isolated from the culturemedium. This method can be applied widely to fine chemicals such asenzymes, vitamins, amino acids, sugars, fatty acids, and natural andsynthetic flavorings, aroma substances and colorants. Especiallypreferred is the production of tocopherols and tocotrienols andcarotenoids. The transformed host organisms are cultured and theproducts are isolated from the host organisms or the culture medium bymethods known to the skilled worker. The production of pharmaceuticalssuch as, for example, antibodies or vaccines, is described by Hood E E,Jilka J M. Curr Opin Biotechnol. 1999 August; 10(4):382-6; Ma J K, VineN D. Curr Top Microbiol Immunol. 1999; 236:275-92.

In one embodiment, the present invention also relates to the use of thepolynucleotide, the vector, or the polypeptide of the present inventionfor producing a plant or a plant tissue, plant organ, or a plant cell ora part thereof resistant to said.

Furthermore, in one embodiment, the present invention relates to amethod for the identification of a compound stimulating resistance to asaid plant pathogen comprising:

-   a) contacting cells which express the polypeptide of the present    invention or its mRNA with a candidate compound under cell    cultivation conditions;-   b) assaying an increase in expression of said polypeptide or said    mRNA;-   c) comparing the expression level to a standard response made in the    absence of said candidate compound; whereby, an increased expression    over the standard indicates that the compound is stimulating    resistance.

Said compound may be chemically synthesized or microbiologicallyproduced and/or comprised in, for example, samples, e.g., cell extractsfrom, e.g., plants, animals or microorganisms, e.g. pathogens.Furthermore, said compound(s) may be known in the art but hitherto notknown to be capable of suppressing or activating Rpi-blb2. The reactionmixture may be a cell free extract or may comprise a cell or tissueculture. Suitable set ups for the method of the invention are known tothe person skilled in the art and are, for example, generally describedin Alberts et al., Molecular Biology of the Cell, third edition (1994),in particular Chapter 17. The compounds may be, e.g., added to thereaction mixture, culture medium, injected into the cell or sprayed ontothe plant.

If a sample containing a compound is identified in the method of theinvention, then it is either possible to isolate the compound from theoriginal sample identified as containing the compound capable ofactivating or increasing resistance to said pathogens, or one canfurther subdivide the original sample, for example, if it consists of aplurality of different compounds, so as to reduce the number ofdifferent substances per sample and repeat the method with thesubdivisions of the original sample. Depending on the complexity of thesamples, the steps described above can be performed several times,preferably until the sample identified according to the method of theinvention only comprises a limited number of or only one substance(s).Preferably said sample comprises substances of similar chemical and/orphysical properties, and most preferably said substances are identical.Preferably, the compound identified according to the above-describedmethod or its derivative is further formulated in a form suitable forthe application in plant breeding or plant cell and tissue culture.

The compounds which can be tested and identified according to a methodof the invention may be expression libraries, e.g., cDNA expressionlibraries, peptides, proteins, nucleic acids, antibodies, small organiccompounds, hormones, peptidomimetics, PNAs or the like (Milner, NatureMedicine 1 (1995), 879-880; Hupp, Cell 83 (1995), 237-245; Gibbs, Cell79 (1994), 193-198 and references cited supra). Said compounds can alsobe functional derivatives or analogues of known inhibitors oractivators. Methods for the preparation of chemical derivatives andanalogues are well known to those skilled in the art and are describedin, for example, Beilstein, Handbook of Organic Chemistry, Springeredition New York Inc., 175 Fifth Avenue, New York, N.Y. 10010 U.S.A. andOrganic Synthesis, Wiley, New York, USA. Furthermore, said derivativesand analogues can be tested for their effects according to methods knownin the art. Furthermore, peptidomimetics and/or computer aided design ofappropriate derivatives and analogues can be used, for example,according to the methods described above. The cell or tissue that may beemployed in the method of the invention preferably is a host cell, plantcell or plant tissue of the invention described in the embodimentshereinbefore.

Determining whether a compound is capable of suppressing or activatingsaid resistance can be done, as described in the examples, in particularvia sporulation index determination. The activator identified by theabove-described method may prove useful as a fungicide or cropprotectant. Thus, in a further embodiment the invention relates to acompound obtained or identified according to the method of the inventionsaid compound being an agonist of Rpi-blb2.

Accordingly, in one embodiment, the present invention further relates toa compound identified by the method of the present invention.

Said compound is, for example, a homologue of Rpi-blb2. Homologues ofthe polypeptid of the present invention can be generated by mutagenesis,e.g., discrete point mutation or truncation of Rpi-blb2. As used herein,the term “homologue” refers to a variant form of the protein which actsas an agonist of the activity of the Rpi-blb2. An agonist of saidprotein can retain substantially the same, or a subset, of thebiological activities of Rpi-blb2.

In one embodiment, the invention relates to an antibody specificallyrecognizing the compound of the present invention.

The invention also relates to a diagnostic composition comprising atleast one of the aforementioned polynucleotides, nucleic acid molecules,vectors, proteins, antibodies or compounds of the invention andoptionally suitable means for detection.

The diagnostic composition of the present invention is suitable for theisolation of mRNA from a cell and contacting the mRNA so obtained with aprobe comprising a nucleic acid probe as described above underhybridising conditions, detecting the presence of mRNA hybridised to theprobe, and thereby detecting the expression of the protein in the cell.Further methods of detecting the presence of a protein according to thepresent invention comprises immunotechniques well known in the art, forexample enzyme linked immunosorbent assay. Furthermore, it is possibleto use the nucleic acid molecules according to the invention inparticular the markers described in the examples, e.g. in table 3a or 3bas molecular markers or primer in plant breeding.

Suitable means for detection are well known to a person skilled in theart, e.g. buffers and solutions for hybridisation assays, e.g. theaforementioned solutions and buffers, further and means for Southern-,Western-, Northern- etc. -blots, as e.g. described in Sambrook et al.are known.

In another embodiment, the present invention relates to a kit comprisingthe polynucleotide, the vector, the host cell, the polypeptide, theantisense nucleic acid, the antibody, plant cell, the plant or planttissue, the harvestable part, the propagation material or the compoundof the invention.

The compounds of the kit of the present invention may be packaged incontainers such as vials, optionally with/in buffers and/or solution. Ifappropriate, one or more of said components may be packaged in one andthe same container. Additionally or alternatively, one or more of saidcomponents may be adsorbed to a solid support as, e.g. a nitrocellulosefilter, a glass plate, a chip, or a nylon membrane or to the well of amicrotiterplate. The kit can be used for any of the herein describedmethods and embodiments, e.g. for the production of the host cells,transgenic plants, pharmaceutical compositions, detection of homologoussequences, identification of antagonists or agonists, etc.

Further, the kit can comprise instructions for the use of the kit forany of said embodiments, in particular for its use for increasing theresistance to one or more of said pathogens of a plant cell, planttissue or plant.

In a preferred embodiment said kit comprises further a polynucleotideencoding one or more of the aforementioned resistance protein(s),preferably Rpi-blb, and/or an antibody, a vector, a host cell, anantisense nucleic acid, a plant cell or plant tissue and/or a plantrelated to said resistance protein(s), preferably to Rpi-blb.

In a further embodiment, the present invention relates a method for theproduction of a crop protectant providing the polynucleotide, the vectoror the polypeptide of the invention or comprising the steps of themethod of the invention; and formulating the polynucleotide, the vectoror the polypeptide of the invention or the compound identified in step(c) of said method in a form applicable as plant agriculturalcomposition.

In another embodiment, the present invention relates to a method for theproduction of a crop protectant composition comprising the steps of themethod of the present invention; and

-   (a) formulating the compound identified in step (c) in a form    acceptable as agricultural composition.

Under “acceptable as agricultural composition” is understood, that sucha composition is in agreement with the laws regulating the content offungicides, plant nutrients, herbicides, etc. Preferably such acomposition is without any harm for the protected plants and the animals(humans included) fed therewith.

The present invention also pertains to several embodiments relating tofurther uses and methods. The polynucleotide, polypeptide, proteinhomologues, fusion proteins, primers, vectors, host cells, describedherein can be used in one or more of the following methods:identification of plants resistant to plant pathogens as mentioned andrelated organisms; mapping of genomes; identification and localizationof sequences of interest; evolutionary studies; determination of regionsrequired for function; modulation of an activity.

Accordingly, the polynucleotides of the present invention have a varietyof uses. First, they may be used to identify an organism as being S.bulbocastanum or a close relative thereof. Also, they may be used toidentify the presence of S. bulbocastanum or a relative thereof in amixed population of plants. By probing the extracted genomic DNA of aculture of a unique or mixed population of plants under stringentconditions with a probe spanning a region of the gene of the presentinvention which is unique to this S. bulbocastanum, one can ascertainwhether the present invention has been used or whether S. bulbocastanumor a relative, e.g. a close relative, is present.

Further, the polynucleotide of the invention may be sufficientlyhomologous to the sequences of related species such that these nucleicacid molecules may serve as markers for the construction of a genomicmap in related organism.

The polynucleotides of the invention are also useful for evolutionaryand protein structural studies. By comparing the sequences of theRpi-blb2 of the present invention to those encoding similar enzymes fromother organisms, the evolutionary relatedness of the organisms can beassessed. Similarly, such a comparison permits an assessment of whichregions of the sequence are conserved and which are not, which may aidin determining those regions of the protein which are essential for thefunctioning of the enzyme. This type of determination is of value forprotein engineering studies and may give an indication of what theprotein can tolerate in terms of mutagenesis without losing function.

These and other embodiments are disclosed and encompassed by thedescription and examples of the present invention. Further literatureconcerning any one of the methods, uses and compounds to be employed inaccordance with the present invention may be retrieved from publiclibraries, using for example electronic devices. For example the publicdatabase “Medline” may be utilized which is available on the Internet.Further databases and addresses are known to the person skilled in theart and can also be obtained. An overview of patent information inbiotechnology and a survey of relevant sources of patent informationuseful for retrospective searching and for current awareness is given inBerks, TIBTECH 12 (1994), 352-364.

Tables:

Table 1: Sequences:

TABLE 2 Segregation of resistance in 2851 progeny clones of BC4 mappingpopulations ARG 95-3 and ARP 96-11 in the field trial of 2000 atMarknesse, The Netherlands. Numbers of clones classified as having aresistant, susceptible or unknown phenotype is presented withpercentages in parenthesis. No clones No clones No clones Mapping withsusceptible with resistant with unknown population phenotype phenotypephenotype Totals ARG 95-3 846 (37) 886 (39) 551 (24) 2283 ARP 96-11 256(45) 170 (30) 142 (25) 568 Totals 1102 (39)  1056 (37)  693 (24) 2851

TABLE 3A Overview of markers used for mapping Rpiblb2 SEQ ID AnnealingRestriction Marker Ori¹⁾ Sequence NO: temp (° C.) Enzyme²⁾ E46M52 FTTGTGGTTATCGATGAGAAT 11 56.5 SCAR (b) R GAAACAACAGCAGGATAGTGAG 12E46M52e F TTGTGGTTATCGATGAGAAT 13 61 SCAR (a, b); RGAAACAACAGCAGGATAGTGAG 14 MboI (c) E40M58 F GAATTCAGCACAAATACCAA 15 50DdeI (a) R TTAACGTTTACTATCACGAG 16 E40M58e F GTAGAAACAGCAGCCTCATAAGC 1755 SCAR (a) R TTCTGCCTAATTGCCCTGTG 18 S1E00 F GGGGTTGGGAAGACAACGACAC 1950 AFLP R AATTCCAAGATACAGTCAAATAC 20 41L F AGGCAGGATTAACAGTAGAAG 21 58TaqI (a) R CATGCTTTTAGGAAGAAGCTC 22 36L F TTGAGACAAAGCAGCTCCAC 23 59ApoI (a, b) R ACGTTTCTCACACCTACAGG 24 69L F TGATGGCACGTTTGATCGTG 25 61TaqI (a, b); R TAAGATCCAAACCAGCCACC 26 HpaII (c) 69R FCCTTATCACACATGTGGCTAC 27 58 RsaI (a, b); R ATTGAAACGGAGGAAGTACAAC 28ApoI (c) 141R F TTCTTCATATGGCAGACCAAC 29 60 RsaI (a, b); RCTACTCTGCTGACATGCAGG 30 DdeI (c) 24L F GAGATTCTCAAAGGTGTCTTCC 31 60 SCAR(a, b, c) R AACCTGTGCTTTCCCATTCG 32 24R F CTTTCACAAGCGTCACTTTGG 33 58SCAR (a, b) R TAAAAAGAATCAACAGGGCAAC 34 14L F ACGACTGCTCAAAGTTGGCC 35 58SCAR (a, b, c) R CCAAGAAGCCAGTTGAGAGC 36 123L F GTAGATTACACTATGGATATGG37 60 SCAR (a, b) R CAGTTAGCAGCAATGTCAGC 38 123L2 FCATTCAACTAGGCCAAAAGTGG 39 59 SCAR (a, b); R CCAGGTAGGTGTTTTCTTCC 40 DraI(c) 123R F GTTCTAAGTCAGATGCCACC 41 62 SCAR (a, b) R AAGTGCTCCAACACGAGCC42 133R F TGAGTTCTCTTACCCTGCG 43 60 SCAR (a, b) R GGATATCCAGCATCAATGCC44 133R2 F GGTGAGCCTCCTTGCATTCC 45 60 SCAR (a, b) R CCTGAGGGAAGATGTCACG46 99L F CCTAGTTTAGAGTGAGTAGAC 47 58 SCAR (a, b) RGTGATATATTGCTCAAGGATCC 48 113R F GTTGCTGGCTGTCACTGATC 49 59 SCAR (a, b)R GTGATGTGCAGGGTTCAAGG 50 67L F GATTAGTGTAGATCTTAGCTTG 51 62 MboI (a, b)R AAATCTCTCTCACAATTATCCC 52 112L F CTATTGACTGAACCTGCTGAG 53 56 HaeIII(a); R TGAAGTCATTTAGTCCACAGC 54 HinfI (c) CT216 F AGATCGGAGTGTGAACATGG55 56 (RFLP) R CTTCTACTTCTAGTCGACTGC 56 CT216 F CGTAGTCCATCTGAAGCTCC 5765 SCAR (a, b) R TCTTCTTCTGCTAGTCGTCG 58 CT119 F ACTATTCTCACGTAAGGGGACAC59 60 HindIII (a, b) R GTGTACATGTATGAAACTCTAGC 60 CT119N FGTTCCTTTCAATCAGAAAGTAG 61 55 SCAR (a) R CTTTGGATGAGTCAAAAGGCT 62 14L24LF univ14L 60 CfoI (c) R univ24L SPB30L F CAAGTTACGGCAACCAAGAG 63 57HpaII (c) R CTTTGACACAGTGTTAGAATGC 64 SPB39L F CGTGATCTAGGAGTTACGAC 6552 SCAR (c) R CTTATTTTAAATACAAGACATCTGG 66 24L9spec F univ. 14L 56 HhaI(c) R CAGAGGAAAGTCAACCAACG 67 24Lspec F univ. 14L 60 CfoI (c) RCAGAGGAAAGTCAACCAACG 68 NptII F TCGGCTATGACTGGGCACAACAGA 69 70 RAAGAAGGCGATAGAAGGCGATGCG 70 M13 F TGTAAAACGACGGCCAGT 71 55 RGGAAACAGCTATGACCATG 72 ¹⁾Ori: Orientation of the primer; F: forwardprimer; R: reverse primers ²⁾a: ARG95-3, b: ARP96-11, c: B6a

TABLE 3B Overview of primers used for mapping Rpi-blb2 primer OriSequence¹⁾ SEQ ID NO: ARO 73 F TTCAGCACAAATACCAAT 73 ARO 74 RGATGTTCCCCTTCTTTTA 74 ARO 77 R TTGTGGTTATCGATGAGAAT 75 ARO 79 RACCTGGCGTTCCTTATTTTT 76 ARO 94 NGTCASWGANAWGAA 77 ARO 128 FGATGGAGCGGAAAAGCCGGTG 78 ARO 129 F GGTGTTTTGTAGCATCTCCAG 79 ARO 295CCATGATTACGCCAAGCTGG 80 ARO 296 GGTTTTCCCAGTCACGACGT 81 univ14L FAGAAAGCTCACCAGTGGACC 82 univ24L R ATTTATGGCTGCAGAGGACC 83 123Mi RAAGTCCAATTGCTCATCCATC 84 14L2 R TGCACCATGCACGAAGGTC 85 24L2 FCAATWTTGGTTCCCGAAATTGG 86 ARF1F F ATGGAAAAACGAAAAGATAATGAAG 87 ARF1R RCTACTTAAATAACGGGATATCCTTC 88 ARO 602 F CCCATGACTCCTTGAGTTTG 89 S1GGTGGGGTTGGGAAGACAACG 90 EcoR1 + 0 GTAGACTGCGTACCAATTC 91 MseI + 0GATGAGTCCTGAGTAA 92 ARO 769 GTGCTTCATTCAAACTCAAGGAG 95 ARO 770CTGAACTAGAAAAACTCACTGTAGA 96 ARO 771 GTTTGAAAAGATTGCAATTGCATG 97 ARO 772CTCAGCCATCAGTTGAAACAGAGA 98 ARO 774 GAGAGAGATTCAAGAGGAGGAAGC 99 ¹⁾N =A + T + G + C, S = G + C, W = A + T

TABLE 4 Complementation of late blight susceptibility in potato cvImpala cv Kondor RGC-containing R plants/ RGC-containing R plants/Source plants/ RGC-containing plants/ RGC-containing BAC-library BACGenotype¹ transformants plants transformants plants ARD 1197-16  24 R₀(RGC1) 12/15^(a)  0/12  8/10^(b) 0/8  24 R₀ (RGC2)  8/11^(a) 0/8 5/6^(b) 0/5  24 R₀ (RGC3) 11/13^(a)  0/11  5/7^(b) 0/5 211 R₀ (RGC4) 5/7^(a) 0/5 10/12^(a)  0/10 242 R₀ (RGC4)  5/7^(a) 0/5 8/8^(a) 0/8 211R₀ (RGC5)  5/7^(a) 4/5 12/13^(a) 12/12 211 R₀ (RGC6) — — — — 211 R₀(RGC24L) — — — BIb 2002 SPB39 R₀ (RGC4)  5/6^(a) 0/5 3/3^(a) 0/3 SPB39R₀ (RGC5) 11/15^(a) 11/11 8/8^(a) 7/8 SPB39 R₀ (RGC6)  3/3^(a) 0/36/6^(a) 0/6 SPB30 R₀ (RGC7)  3/4^(a) 0/3 9/9^(a) 0/9 SPB30 R₀ (RGC8) 1/1^(a) 0/1 — — SPB39 R₀ (24L) — — R₀ (pBINPLUS)  3/3 0/3  8/10 0/8 ¹R₀genotypes are primary transformants obtained from transformation of thesusceptible potato cultivars Impala or Kondor with T-DNA constructscontaining the Rpi-blb2 gene candidates RGC1 to RGC8 and RGC24L or anempty pBINPLUS vector. Agrobacterium tumefaciens strains UIA143^(a) orAGL0^(b) were used for transformation of the P. infestans susceptiblepotato cultivars Impala and Kondor.

TABLE 5 Cycling conditions used for TAIL-PCR Reaction cycle no. Thermalcondition Primary 1 92° C. (2 min), 95° C. (1 min) 5 94° C. (15 s), 63°C. (1 min), 72° C. (2 min) 1 94° C. (15 s), 30° C. (3 min), ramping to72° C. over 3 min, 72° C. (2 min) 10  94° C. (5 s), 44° C. (1 min), 72°C. (2 min) 12^(a ) 94° C. (5 s), 63° C. (1 min), 72° C. (2 min) 94° C.(5 s), 63° C. (1 min), 72° C. (2 min) 94° C. (5 s), 44° C. (1 min), 72°C. (2 min) 1 72° C. (5 min) Secondary 10^(a ) 94° C. (5 s), 63° C. (1min), 72° C. (2 min) 94° C. (5 s), 63° C. (1 min), 72° C. (2 min) 94° C.(5 s); 44° C. (1 min), 72° C. (2 min) 1 72° C. (5 min) Tertiary 20  94°C. (10 s), 44° C. (1 min), 72° C. (2 min) 1 72° C. (5 min) ^(a)these arenine-segment super cycles each consisting of two high-stringency and onereduced-stringency cycle

TABLE 6 Complementation of late blight susceptibility in tomato cultivarMoneyMaker by Rpi-blb2 RGC-containing R plants/ Bac- Source plants/ RGC-library Bac genotype transformants containing plants Blb 2002 SPB39 R₀(RGC5) 24/25 22/24 R₀ genotypes are primary transformants obtained fromtransformation of the susceptible tomato cultivar Moneymaker with theT-DNA construct containing the Rpi-blb2 gene RGC5. Agrobacteriumtumefaciens strains UIA143^(a) was used for transformation of the P.infestans susceptible tomato cultivar.

The figures show:

FIG. 1. Schematic representation of the development of the complexinterspecific hybrid clones designated as ‘ABPT’ (1 a) and the S.tuberosum mapping populations that were derived from two of theseclones: ABPT clone 55 and ABPT clone 60 (1 b to d). A; Solanum acaule,B; S. bulbocastanum, P; S. pureja, T; S. tubersosum, 2x; diploid(2n=2x=24), 3x; triploid, 4x; tetraploid, 6x; hexaploid, cv; cultivar.Codes in italics indicate mapping populations.

FIG. 2. Disease progress curves for clone ARF 87-601 and susceptiblecontrol cultivars (cv) Bildtstar, Eersteling and the partial resistantcontrol cultivar Pimpernel in a field test for foliar resistance to lateblight in Toluca Valley, Mexico in 1991. At eight time points afterplanting, the percentage-blighted foliage due to a natural late blightinfection was scored on the 1 to 9 CIP scale (Estrada-Ramos, 1983).

FIG. 3. Disease progress curves for clone ARF 87-507, ARF 87-601, ARF87-801, the susceptible control cultivar (cv) Granola and the partialresistant breeding clone AR 85-96-13 in a field test for foliarresistance to late blight in Benguet Province, Philippines in 1992. Atsix time points between August 25^(th) to November 24^(th), thepercentage-blighted foliage due to a natural late blight infection wasscored on the 1 to 9 CIP scale (Estrada-Ramos, 1983).

FIG. 4. Typical phenotypes in tetraploid resistant and susceptibleparental clones and progeny clones segregating for Rpi-blb2 mediatedresistance to late blight in the annual field trial at Marknesse, TheNetherlands, approximately 6 weeks after inoculation with isolateIPO82001 of P. infestans. Six plant plots with a clone showing theresistant phenotype (within black solid line) that shows no or hardlyany sporulating lesions and with a clone showing the susceptiblephenotype (within white dotted line) that shows completely blightedfoliage.

FIG. 5. Genetic map based on 109 progeny clones of S. tuberosum mappingpopulation ARG 95-15 showing 7 AFLP markers that were found tocosegregate with the Rpi-blb2 locus. Numbers left to the vertical lineindicate the genetic distance between flanking markers or the Rpi-blb2locus in centimorgan (cM).

FIG. 6. Genetic map based on 137 progeny clones of S. tuberosum mappingpopulation ARG 95-3 showing 15 AFLP markers and RGA marker S1E00 thatwere found to cosegregate with the Rpi-blb2 locus. Phenotypes of theprogeny clones were obtained with detached leaf assays. Numbers left tothe vertical line indicate the genetic distance between flanking markersor the Rpi-blb2 locus in centimorgan (cM).

FIG. 7. Genetic map based on 178 progeny clones of S. tuberosum mappingpopulation ARG 95-3 showing 5 markers that were found to cosegregatewith the Rpi-blb2 locus on linkage group 6 of S. tuberosum. Phenotypesof the progeny clones were determined in the field trial at Marknesse,the Netherlands in 1998. Markers E40M58 and E46M52 were scored either asAFLP, CAPS, SCAR or extended (suffix: e) marker (table 3A). Partly,marker CT119 was scored as marker CT119N (table 3a). Marker CT216 wasscored as SCAR marker. The number left to the vertical line indicatesthe genetic distance between flanking markers or the Rpi-blb2 locus incentimorgan (cM). For each marker, the number of recombinants betweenmarker and phenotype and the total number of progeny clones scored isgiven in parenthesis.

FIG. 8. Genetic maps based on 886 progeny clones of S. tuberosum mappingpopulation ARG 95-3 and on 170 progeny clones of S. tuberosum mappingpopulation ARP 96-11, showing markers that were found to cosegregatewith the Rpi-blb2 locus on linkage group 6 of S. tuberosum. Phenotypesof the progeny clones were determined in the field trial at Marknesse,the Netherlands in 2000. The number left to the vertical line indicatesthe genetic distance between flanking markers in centimorgan (cM). Themarker interval which delimitates the position of the Rpi-blb2 gene,based on detected recombination events in progeny clones, is indicatedby double arrow headed lines.

FIG. 9. Physical map of the genomic region containing Rpi-blb2 in S.tuberosum (upper horizontal line) and S. bulbocastanum (lower horizontalline). Vertical lines indicate the relative position of markers linkedto resistance. Numbers above the horizontal lines are the number ofrecombinants identified between the flanking markers in 1056 and 1899progeny plants of S. tuberosum, derived from complex species hybrids“ABPT” (FIG. 1), and S. bulbocastanum progeny plants respectively.ABPT-derived progeny comprises clones from both the mapping populationsARG 95-3 and ARP 96-11. Rectangles represent bacterial artificialchromosome (BAC) clones from the ARD 1197-16 BAC library except for BACclones with prefix “Blb” which were from the S. bulbocastanum Blb 2002BAC library. The marker interval which delimitates the position of theRpi-blb2 gene, based on detected recombination events in progeny clones,is indicated by double arrow headed lines. Small arrows indicatepositions of Resistance Gene Candidates (RGC's).

FIG. 10. Schematic representation of the development of the diploid,intraspecifc mapping population B6 of S. bulbocastanum. Codes in italicsindicate mapping populations.

FIG. 11. Genetic map based on 1899 progeny clones of S. bulbocastanummapping population B6, showing markers that were found to cosegregatewith the Rpi-blb2 locus on chromosome 6 of S. bulbocastanum. Phenotypesof the progeny clones were determined by detached leaf assays. Thenumber left to the vertical line indicates the genetic distance betweenflanking markers in centimorgan (cM). The marker interval whichdelimitates the position of the Rpi-blb2 gene, based on detectedrecombination events in progeny clones, is indicated by a double arrowheaded line.

FIG. 12. Genetic complementation for late blight susceptibility. Typicaldisease phenotypes of potato (S. tuberosum) leaves, 6 days afterinoculation with a sporangiospore suspensions of P. infestans isolate655-2A. Leaf derived from kanamycin resistant cv Kondor plantstransformed with pBINPLUS (control; A), leaves derived from cv Kondorplants harbouring BAC SPB39 derived (B) or BAC 211 derived RGC5 (C),leaf derived from kanamycin resistant cv Impala plants transformed withpBINPLUS (control; D), leaves derived from cv Impala plants harbouringBAC SPB39 derived (E) or BAC 211 derived RGC5 (F). Panels A and D depicttypical susceptible responses with extensive sporulating lesions of P.infestans. Panels B, C, E and F depict typical resistance reactionsobserved at the sites of inoculation on transgenic potato plantsharbouring Rpi-blb2.

FIG. 13. Nucleic acid sequences coding for the Rpi-blb2 gene. A. Codingnucleic acid sequence of the Rpi-blb2 gene (SEQ ID NO: 1). B. Codingnucleic acid sequence of the Rpi-blb2 gene including the intron sequence(position 43-128) (SEQ ID NO: 3). C. Sequence of the 7967 bp Sau3AIgenomic DNA fragment of ARD 1197-16 BAC 211 present in p211F-C12 (SEQ IDNO: 5), one of the two genetic constructs used for geneticcomplementation for late blight resistance. The genomic fragmentharbours the Rpi-blb2 gene including natural regulatory elementsnecessary for correct expression of the gene. The initiation codon (ATGposition 1546-1548) and the termination codon (TAG position 5433-5435)are underlined. D. Sequence of the 9949 bp Sau3AI genomic DNA fragmentof S. bulbocastanum 2002 BAC BlbSP39 present in pSP39-20 (SEQ ID NO: 6),one of the two genetic constructs used for genetic complementation forlate blight resistance. The genomic fragment harbours the Rpi-blb2 geneincluding natural regulatory elements necessary for correct expressionof the gene. The initiation codon (ATG position 1413-1415) and thetermination codon (TAG position 5300-5303) are underlined.

FIG. 14. Putative Rpi-blb2 gene structure and deduced Rpi-blb2 proteinsequence. A. Schematic representation of the Rpi-blb2 gene structure.Horizontal lines indicate exons. Open boxes represent coding sequence.Lines angled downwards indicate the positions of intron sequences. B.Deduced Rpi-blb2 protein sequence (SEQ ID NO: 4). The amino acidsequence deduced from the DNA sequence of Rpi-blb2 is divided into threedomains (LZ, NBS and LRR). Hydrophobic residues in domain A that formthe first residue of heptad repeats of the potential leucine zipper (LZ)domain are underlined. Conserved motifs in R proteins are written inlowercase and in italic in the NBS domain. Residues matching theconsensus of the cytoplasmic LRR are indicated in bold in the LRRdomain. Dots in the sequence have been introduced to align the sequenceto the consensus LRR sequence of cytoplasmic LRRs.

FIG. 15. Alignment of the deduced protein products encoded by Rpi-blb2(SEQ ID NO: 4), Mi-1.1 (SEQ ID NO: 8) and Mi-1.2 (SEQ ID NO: 10). Thecomplete amino acid sequence of Rpi-blb2 is shown and amino acidresidues from Mi-1.1 and Mi-1.2 that differ from the correspondingresidue in Rpi-blb2. Dashes indicate gaps inserted to maintain optimalalignment. Amino acid residues that are specific for Rpi-blb2, whencompared to those at corresponding positions in Mi-1.1 and Mi-1.2 arehighlighted in bold and red. The regions of the LRRs that correspond tothe β-strand/β-turn motif xxLxLxxxx are underlined. Conserved motifs inthe NBS domain are indicated in lowercase. A vertical line indicates thedivision between CC-NBS and LRR region. The position of the VLDL motifwhich is conserved in the third LRR of many plant R proteins but not inRpi-blb2 is indicated by a shaded rectangle.

FIG. 16. CLUSTAL W (1.82) Multiple Sequence Alignments of Mi1.1 (SEQ IDNO: 7), Mi1.2 (SEQ ID NO: 9) and Rpi-blb2 (SEQ ID NO: 1) nucleic acids.

FIG. 17. CLUSTAL W (1.82) Multiple Sequence Alignments of Mi1.1 (SEQ IDNO: 8), Mi1.2 (SEQ ID NO: 10) and Rpi-blb2 (SEQ ID NO: 2) proteins.

FIG. 18. Typical phenotypes of the resistance genes R2 (A) and Rpi-blb2(B) compared to a susceptible phenotype of cv. Bintje (C). Panel Adepicts a typical hypersensitive response reaction with very smallnecrotic spots, while panel B shows large necrotic regions that containa low level of sporulation. Panel C depicts a typical susceptiblereaction with clear sporulation.

This invention is further illustrated by the following examples whichshould not be construed as limiting.

EXAMPLES Example 1 Evaluation of Resistance in ABPT Derived Back CrossClones and Populations

BC2-clones ARF 87-507 and ARF 87-801 were selected from BC2-progenyobtained after two rounds of backcrossing on complex species hybridABPT-clone number 55 (FIG. 1 a) with late blight (LB) susceptible S.tuberosum cultivar Oberambacher Frühe as first parent and S. tuberosumcultivars Arkula (FIG. 1 b) and Blanka (FIG. 1 c) respectively as secondparents. Similarly, BC2-clone ARF 87-601 was obtained by successivecrossing on ABPT-clone 60 with LB susceptible S. tuberosum cultivarsAlcmaria and Blanka (FIG. 1 d).

Clone ARF 87-601 was tested as part of a field test for screening ofLB-resistance in the Toluca area in Mexico in 1991. A plot of clone ARF87-601 with seven plants was evaluated in comparison to plots with nineplants each of the control cultivars Bildtstar, Eersteling andPimpernel. According to the ratings for resistance to late blight in theDutch National list of recommended potato cultivars of 1988, thesecontrol cultivars scored 3, 3 and 8 respectively on a scale from 3 to 8of increasing resistance. Cultivar Pimpernel is considered as a sourceof partial resistance (Colon et al., 1985). About forty days afterplanting, natural infection by P. infestans established. The developmentof LB in the foliage then was monitored eight times during the periodfrom July 16^(th) to September 2^(nd) (FIG. 2). There was a cleardifference between the disease progress curves for ARF 87-601 incomparison to the control cultivars. At 74 days after planting, foliageof the control cultivars was completely or nearly completely blightedwhereas clone ARF 87-601 showed no visible symptoms (FIG. 2). Clones ARF87-507, ARF 87-801 and again clone ARF 87-601 showed comparable resultsin a field test for screening of LB-resistance in the Benguet Provinceof the Philippines in 1992 (FIG. 3). Ten plants each of the three BC2clones, control cultivar Granola and the moderately LB resistantbreeding clone AR 85-96-13, which was used as female parent to obtain AR92-1197 (FIG. 1 d), were planted on August 25^(th). The percentage ofblighted foliage was scored six times after occurrence of naturalinfection by P. infestans. Disease progress curves of ABPT derivedBC2-clones were markedly different when compared to cultivar Granola andclone AR 85-96-13 (FIG. 3). BC2-clones showed no or little LB symptomsand no clear disease progress during the scoring period whereas cultivarGranola had almost completely blighted foliage at the third scoringdate.

Clones ARF 87-601, ARF 87-507 and ARF 87-801 were used for furtherbackcrossing with LB susceptible cultivars and breeding clones of S.tuberosum (FIG. 1 b to 1 d). This breeding work resulted in fourdifferent mapping populations, tetraploid BC3-population ARG 95-15,tetraploid BC4-populations ARG 95-3 and ARP 96-11 and diploidBC4-population DP1. During the successive steps of this breeding workresistant clones ARF 87-507, ARF 87-601, ARF 87-801, AR 91-1263, AR91-1292 and AR 92-1197 were selected on the basis of agronomicperformance in common practice breeding evaluations as well as byscreening their parents and relevant progenies in a field trial atMarknesse, the Netherlands, that was inoculated with the complex isolateIPO82001 of P. infestans. The diploid (2n=2x=24) clone ARD 1197-16 wasselected among the progeny of cross AR 92-1197×Phu 81-101 (FIG. 1 d),the latter parental clone being known for its capacity to induceparthenogenic seed set in the female parent (Hermsen and Verdenius,1973). Initially, resistance to LB in ARD 1197-16 was found afterrepeated detached leaf assays using P. infestans isolates IPO82001,IPO655-2A and IPO428-2 and verified in a field trial in 1998 atMarknesse. The diploid status of clone ARD 1197-16 was confirmed by flowcytometry (Plant cytometry services, Schijndel, the Netherlands).

Clear segregation for the LB-resistance trait in ABPT-derived progenyand mapping populations was observed during successive years of fieldtesting at the trial site of Marknesse, approximately 6 weeks afterinoculation with isolate IPO82001 of P. infestans. Typically, resistantclones showed no or hardly any sporulating lesions whereas susceptibleclones showed completely blighted foliage (FIG. 4) In 2000, a total of2851 clones from the mapping populations ARG 95-3 and ARP 96-11 werescreened as single plant plots. On average, 24 percent of the clonesshowed phenotypes that could not unambiguously be classified asresistant or susceptible. Clones that could be classified as such showedsegregation ratio's of resistant to susceptible phenotypes of 1 to 1 and1 to 1.5 for populations ARG 95-3 and ARP 96-11, respectively (Table 2).

Detached leaf assays with ABPT-derived progeny and mapping populationswhere found to be less accurate for phenotyping than screening underfield conditions. Nevertheless, results of detached leaf assays wereconsidered suitable for the initial determination of the phenotype ofindividual clones and thus, for construction of mapping populations.

Example 2 Genetic Mapping of the Rpi-blb2 Resistance Locus in ABPTDerived Back Cross Populations

In all four mapping populations (FIG. 1), resistance segregated asexpected for a monogenic trait, suggesting the presence of a dominantresistance allele at a single locus (Table 2). This locus was designatedthe Rpi-blb2 locus.

In order to identify markers linked to Rpi-blb2, an initial AFLPanalysis with 14 primer combinations (pc) was carried out on DNA of 10resistant and 10 susceptible ARG 95-15 progeny plants, based on detachedleaf assay, including the parental clones. The testing of 21 potentiallylinked markers on an additional 89 plants identified several markerslinked to resistance (FIG. 5). Subsequent bulked segregant analysis(BSA) with 160 pc's on 2 resistant and 2 susceptible DNA pools, eachcontaining genomic DNA of 8 resistant or susceptible ARG 95-15 progenyplants, respectively, identified a total of 58 AFLP markers potentiallylinked to resistance (FIG. 5). When a number of these markers weretested on 137 progeny plants of ARG 95-3, they were also linked toresistance in this population, suggesting that the resistance in the twopopulations was determined by the same locus (FIG. 6). Thesecosegregating markers mapped 3 to 28 centimorgan (cM) and 1 to 7.2 cM toone side of the locus in ARG 95-15 and ARG 95-3 respectively, suggestingthat Rpi-blb2 could be situated at a distal position on a chromosome.

To determine the position of the Rpi-blb2 on the genetic map of potato,the two cosegregating AFLP markers E40M58 and E46M52 (FIG. 6) werecloned into the pGEM-T vector (Promega, the Netherlands) and sequenced.Primers designed on the ends of the sequences of the cloned AFLPfragments (Table 3) were used to develop cleaved amplified polymorphicsequence (CAPS) marker E40M58 that was found to be cosegregating withthe resistance trait in 25 resistant and 25 susceptible clones of ARG95-3. CAPS marker E40M58 was subsequently tested on 46 progeny plants ofthe C×E mapping population (van Eck et al., 1995). These data were addedto the existing marker scores of the C×E population. Joinmap (Stam,1993) linkage analyses mapped E40M58 8 cM distal to GP79 (Gebhardt etal., 1991), positioning Rpi-blb2 on the short arm of chromosome 6. In178 progeny plants of population ARG 95-3 no recombination betweenRpi-blb2 and AFLP markers E40M58, E40M60 and CAPS marker CT119 wasobserved. AFLP marker E46M52 and sequence characterised amplified region(SCAR) marker CT216 mapped 2.2 cM proximal to the gene (FIG. 7).

Example 3 Identification of a RGA Marker Linked to Rpi-blb2

In an attempt to identify functionally relevant markers linked toresistance, primers designed on the conserved motifs of the NBS domainof plant R genes (Leister et al., 1996), were used in an adapted AFLPprotocol (RGA-AFLP) to identify resistance gene analogue (RGA) specificmarkers.

Using the P-loop based primer S1 from Leister et al. (1996) incombination with the Eco00 AFLP primer, an RGA specific marker, S1E00was developed which cosegregated with resistance and markers E40M58 andCT119 in the ARG 95-3 mapping population (FIGS. 6 and 7).

Example 4 Development of E40M58e and E46M52e SCAR Markers forRecombinant Screening

Using genomic DNA of AR 91-1263 as template, the cloned fragment of AFLPmarker E46M52 was extended by TAIL-PCR. The primary TAIL-PCR wasperformed using primers ARO 77 (sp1) and ARO 94 (AD) Subsequently, thesecondary PCR was performed using ARO 128 (sp2) and the tertiary PCRusing ARO 129 (sp3) both in combination with primer AD. This resulted inan E46M52e fragment that was extended on the 5′ end with approximately500 bp. The E46M52e fragment was cloned in pGEM-T and sequenced. A newforward primer was designed on this sequence and PCR in combination withprimer ARO 77 resulted in SCAR marker E46M52e that cosegregated with theresistant phenotype in the four S. tuberosum mapping populations and asCAPS marker also in population B6.

Using genomic DNA of ARD 1197-16 as template, the cloned fragment ofAFLP marker E40M58 was also extended by TAIL-PCR. The primary TAIL-PCRwas performed in both the 5′ and 3′ directions using sp1 primers ARO 73(3′) and 74 (5′) in combination with primer AD. Subsequently, thesecondary PCR was performed using as sp2 ARO 82 or 79, respectively. Thefragments obtained from the secondary PCR, 750 bp from the 3′ end and400 bp from the 5′ end were cloned in pGEM-T and sequenced. On the basisof both sequences, two new primers were designed resulting in a SCARmarker that cosegregated with resistance in mapping population ARG 95-3and DP1 (Table 3). The fragment of SCAR marker E40M58e could beamplified in the resistant parents of mapping populations ARG 95-3 andDP1, which were both derived from ABPT clone 55 (FIG. 1), but PCRamplification in the parents or progeny clones of mapping populationsARP 96-11 and ARG 95-15, which were both derived from ABPT clone 60, didnot give any detectable PCR product. It was assumed that this could havebeen caused by minor differences in the genomic sequence and therefore,the AFLP fragment was extended by TAIL-PCR using genomic DNA of clone AR91-1292 as template. A fragment E40M58e2 of approximately 300 bp wasobtained, cloned and sequenced. Comparison of the sequence with theoriginal fragment of AFLP marker E40M58 showed that only the first 37 bpof the extended fragment were identical. PCR with primers designed onthe sequence of E40M58e2 did not result in a polymorphic marker. Both ofthe extended markers E40M48e and E40M58e2 were tested on five resistantor susceptible clones of S. bulbocastanum (BGRC 8005 and 8006). Only thefragment of SCAR marker E40M58e could be amplified in four S.bulbocastanum clones, indicating that part of the sequence of E40M58e2was not derived from S. bulbocastanum. This observation suggested thatE40M58e was located on the border of the S. bulbocastanum introgressionfragment in clone AR 91-1292 and that the position of the Rpi-blb2 locuswas proximal to marker E40M58e.

Example 5 Mapping of Rpi-blb2 in a Diploid Mapping Population Derivedfrom ABPT Material

A total of 149 progeny clones of diploid mapping population DP1 werescreened with markers E40M58e and E46M52e. No recombination was foundbetween these markers suggesting suppressed recombination in the genomicregion studied when compared to the tetraploid mapping population ARG95-3 (FIG. 7). A subset of 112 clones was screened for resistance to P.infestans isolates IPO82001, IPO655-2A and IPO428-2 in a partiallyrepeated detached leaf assay. Eleven of the clones (11%) showedintermediate reactions and were classified as having unknown phenotypes.Another 51 and 50 clones were classified as resistant and susceptiblerespectively. Three progeny clones DP1-28, DP1-79 and DP1-81 wereidentified that were putatively recombined between the Rpi-blb2 locusand the markers E40M58e and E46M52e. In 2000, a subset of 50 out of the112 phenotyped clones was tested for resistance to LB in the field atthe trial site of Marknesse. Conclusive results on the phenotype for LBresistance were obtained for 33 out of the 50 clones. The phenotype ofclones 28 and 81 as determined with the detached leaf assay appeared tobe erroneous. Thus, it was concluded that these clones did not representrecombination events between Rpi-blb2 and the markers used. Thephenotype of clone DP1-79 could not be verified conclusively under fieldconditions and this clone may represent the only recombination eventbetween the Rpi-blb2 locus and the markers E40M58e and E46M52e in 101progeny clones of DP1 (1 cM). Since it was shown that two markers,linked to the resistance trait in ARG 95-15, ARG 95-3 and ARP 96-11,cosegregated with the same locus for LB-resistance in DP1, it wasconcluded that the DP1 parental clone ARD 1197-16 was suitable as asource for Rpi-blb2 gene isolation in a map based cloning approach.

Example 6 Physical Mapping of the ABPT Derived Rpi-blb2 Locus

The resistant clone ARD 1197-16, heterozygous for the Rpi-blb2 locus,was used as source DNA for the construction of a BAC library (hereafterreferred to as the ARD 1197-16 BAC library). High molecular weight DNApreparation and BAC library construction were carried out as describedin Rouppe van der Voort et al. (1999). Initially, a total of 67968clones with an average insert size of 100 kb, which corresponds toapproximately 7 genome equivalents, were individually stored in 177384-well microtiter plates at −80° C. Marker screening of the ARD1197-16 BAC library was carried out as described in Rouppe van der Voortet al. (1999). Essentially, DNA pools generated for each 384-well platewere screened by PCR with SCAR or CAPS markers linked to the Rpi-blb2locus in order to build a BAC contig across the Rpi-blb2 locus.

Screening of the ARD 1197-16 BAC library with markers E40M58e, S1E00 andCT119 identified several positive BAC clones, which served as seed BACsfrom which a chromosome walk across the Rpi-blb2 locus was initiated.Marker E40M58e was used to isolate the BAC clones 69 and 141 whereas BACclones 14, 24, 123 and 133 were positive for marker S1E00. Marker CT119was used to isolate BAC 67. After sequencing the left (L) and right (R)borders of these BAC clones, a new set of markers was developed; 14L,24L, 24R, 69L, 69R, 141R, 123L, 123R, 133R and 67L. Screening of theisolated BAC clones with these markers showed that the following pairsof BAC clones shared overlap: the right side of 123 with the left sideof 133, 14 completely with 24, and the left side of 69 with the rightside of 141. BAC 67 did not share overlap with the other BAC clones. Thefinding that the S1E00 positive BAC clones 14, 24, 123, and 133 did notform a single contig indicated that S1E00 was a repetitive sequence.This, together with the finding that the right BAC-end sequences of BACclones 24 and 123 showed high homology to different regions of the Mi1resistance gene from tomato (Milligan et al., 1998, Simons et al.,1998), suggested that the Rpi-blb2 locus harboured more than one RGA.Screening of the initial ARD 1197-16 BAC library with markers 141R, 24L,24R and 123L did not lead to contig extension. However, screening of thelibrary with markers 123R and 133R resulted in the isolation of BACclones 99 and 113, thereby extending the BAC 123/133 contig in onedirection. BAC-end sequencing of these two BAC clones lead to thedevelopment of two new markers, 99L and 113R. Screening of the ARD1197-16 BAC library with 69R lead to the extension of the 141/69 contig.Consecutive screening of the BAC library with markers derived from BACclones that further extended this contig lead to the isolation of BACclones 36, 41 and 112, and the development of markers 36L, 41 L and112L.

In an attempt to complete the BAC contig across the Rpi-blb2 locus, theARD 1197-16 BAC library was enlarged with an additional 38864 BAC clonesof ˜100 kb (384-well plate numbers 178-273). This second library wasscreened with markers 24L, 24R, 123L, and 141R, leading to theidentification of BAC clones positive for both 24R and 123L (e.g. 191)and BAC clones positive for 24L (211, 242). In this way, the gap betweenBAC 24 and 123 was closed and the 24/14 contig was extended towards BACclone 141. There were no new clones in the extended ARD 1197-16 librarythat were positive for marker 141R.

Example 7 Construction of Additional Markers in BAC 123/133 Region

In an attempt to develop additional polymorphic markers from BAC 123 and133, a 10 kb sub-clone library was constructed of both BAC 123 and 133.BAC DNA was partially cleaved with Sau3AI and fragments of approximately10 kbp were cloned in the BamHI site of vector pBINPLUS. In order toselect clones containing the original BAC-end sequence, 288 subclones ofBAC 123 and 192 of BAC 133 were screened with the BAC-end markers 123Lor 133R. In total 14 subclones were positive for marker 123L and 11 formarker 133R. Subsequently, the orientation of the BAC-end positiveclones was determined by several PCRs using either the forward orreverse primer of the relevant BAC-end marker in combination withprimers M13F or M13R (Table 3). For marker 123L three sub-clones and twosub-clones for marker 133R were selected and the ends not containing the123L or 133R marker were sequenced (approximately 500 bp). Based on thenew sequence two new primers were designed for subclone 123 resulting inmarker 123L2 and two new primers were designed for subclone 133resulting in marker 123R2. SCAR marker 123L2, which was located 10 kbpproximal to marker 123L, appeared to be polymorphic in mappingpopulations ARG 95-3, ARP 96-11 and as CAPS in B6. SCAR marker 133R2,which was located 10 kbp distal to marker 133R, was only polymorphic inmapping populations ARG 95-3 and ARP 96-11.

Example 8 Fine Mapping of the Rpi-blb2 Locus in ABPT Derived MappingPopulations

In order to fine map the Rpi-blb2 locus in ABPT derived mappingpopulations a total of 2283 new progeny clones of mapping population ARG95-3 and 598 clones of mapping population ARP 96-11 were tested forresistance to LB in the field at the trial site of Marknesse in 2000(Table 2). In population ARG 95-3 846 clones (37%) were scoredsusceptible and 886 clones resistant (39%). The phenotypes of theremaining 551 clones were unclear. In population ARP 96-11 256 clones(45%) were scored susceptible and 170 clones (30%) resistant. Thephenotypes of the remaining 142 (25%) were unclear (Table 2). The 846and 170 resistant clones from mapping populations ARG 95-3 and ARP96-11, were selected for recombinant screening with SCAR marker CT216and CAPS marker 41L or 36L, respectively. In total 85 (9.6 cM) and 22(12.9 cM) recombinants were obtained in mapping populations ARG 95-3 andARP 96-11 respectively, that were subsequently screened with CAPS marker67L, reducing the number of recombinants to 5 (0.56 cM) in the markerinterval 67L-36L in case of mapping population ARG 95-3 and to 4recombinants (2.35 cM) in the marker interval 67L-41L in case of themapping population ARP 96-11 (FIG. 8). These remaining 9 recombinantswere further analysed with SCAR and CAPS markers 113R, 99L, 133R, 133R2,123R, 123L, 24R, 14L, 24L, 141R, 69L, E40M58e and 69R. The latter twomarkers were scored only in mapping population ARG 95-3.

In population ARG 95-3 two clones showed recombination between markersE40M58e and 69L, positioning the Rpi-blb2 gene 0.23 cM proximal tomarker E40M58e. Two other clones were recombined between markers 113Rand 67L and one was recombined between markers 133R2 and 133R,positioning the Rpi-blb2 gene 0.11 cM distal to marker 133R.

In population ARP 96-11, no recombination was detected between markers41L and 69L, positioning the Rpi-blb2 gene 0.58 cM proximal to marker36L. Two progeny clones were recombined between markers 113R and 67L,and one clone was recombined between markers 99L and 133R, positioningthe Rpi-blb2 gene 0.58 cM distal to marker 99L (FIG. 8; FIG. 9).

Example 9 Evaluation and Genetic Mapping of Late Blight Resistance in aS. bulbocastanum Intraspecific Mapping Population

In order to develop an intraspecific mapping population of S.bulbocastanum, a resistant clone Blb 2002 was obtained from an interaccession cross (FIG. 10). This clone was reciprocally crossed with asusceptible clone Blb 48-5 that was selected also in progeny from aninter accession cross (FIG. 10). The resulting population was designatedB6 with synonyms B6a, Blb 99-229, Blb 00-7 and Blb 00-8.

Initially a small group of 47 progeny plants of the B6 population wasscreened for resistance to P. infestans in a partially repeated detachedleaf assay using a sporangiospore solution of isolate IPO655-2A of P.infestans as inoculum. Plants with leaves that clearly showedsporulating lesions 6 to 9 days after inoculation were considered tohave a susceptible phenotype whereas plants with leaves showing novisible symptoms or necrosis at the side of inoculation in the absenceof clear sporulation were considered to be resistant. Of the 47seedlings, 23 scored resistant and 24 susceptible. These data indicatedthat the progeny of mapping population B6 gave clear segregation of theresistance trait in the detached leaf assay and that resistance could bedue to a single dominant gene or a tightly linked gene cluster. In orderto determine the chromosome position of this locus, 46 seedlings wereanalysed with markers 112L and E46M52e. Marker 112L was found to belinked in repulsion with the resistant phenotype, as only tworecombinants were obtained between this marker and the phenotype of the46 seedlings (4 cM). Also, marker E46M52e was found to be linked inrepulsion with the resistant phenotype. Here, five recombinants wereobtained between marker E46M52e and the phenotype (11 cM). Furthermore,markers 69R, 69L and 141R were used for analysis of the sevenrecombinants between markers 112L and E40M58e with an additional groupof 6, 15 and 14 non recombined seedlings respectively, and found to becompletely linked in either coupling (marker 69R) or repulsion phase(markers 69L and 141R) to resistance, indicating that the resistancegene was located at the same locus, i.e. Rpi-blb2, as in theABPT-derived mapping populations.

In order to determine the position of Rpi-blb2 more precisely relativeto the available markers, another 849 seedlings of the B6 mappingpopulation and 1054 seedlings from the reciprocal cross (FIG. 10) weregrown and analysed for recombination between the markers E46M52e and112L. Thus, in addition to the initial 47 seedlings, a total of 1903individual offspring clones of the B6 population were screened.Recombination between markers E46M52e and 112L was detected in a totalof 138 of these seedlings (7.25 cM). Fine mapping of the Rpi-blb2 locuswas carried out in two steps. Firstly, the group of 138 recombinants wasreduced to 19 by additional screening with markers 14Lb, 113R, 123L2,24L, 141R and 69L (Table 3), derived from left (L) and right (R) bordersequences of BAC clones isolated from the ARD 1197-16 BAC library andsubsequent selection of all the seedlings that were recombined betweenmarkers 113R and 69L. Possibly due to double recombination, 4recombinants gave patterns for the markers scored that deviated fromscores expected in the case of single recombination events in thegenetic interval studied and when assuming co-linearity of markers.These were withdrawn from further analyses. Secondly, the remaining 15recombinants were analysed with markers from border sequences of BACclones isolated from the Blb 2002 library, SPB39L and SPB30L, or withMiGA markers 24L9spec, 24Lspec and 14L24L (Table 3). Results of markeranalyses of these remaining 15 recombinants, which gave clearlyinterpretable marker scores and phenotypes, positioned the Rpi-blb2locus between markers 69L and 24L, on a 0.11 cM (n=1899) geneticinterval (FIG. 11).

Example 10 MiGA Markers

Southern analysis of BAC clones 14, 24, 123 and 133 using markers 123R,14L, or 24L as probes showed that these BAC clones contained severalresistance gene analogs (RGAs). In view of the homology between thesequences of markers 14L, 24L and 123R with the Mi1 gene from tomato,RGAs within the Rpi-blb2 region are hereafter referred to as Mi geneanalogs (MiGAs). In an attempt to develop additional polymorphic markerswithin the Rpi-blb2 interval, PCR fragments generated from BAC clones 24and 123 with the primer combination 14LR and 24LF were cloned into thepGEM-T vector (Promega, the Netherlands) and partially sequenced. Basedon the alignment of these partial sequences, a set of universal primerswere designed, univ14L and univ24L (Table 3), with the aim to amplifythe corresponding region of as many as possible MiGAs within theRpi-blb2 interval. This universal primer set was subsequently used todevelop MiGA specific SCAR/CAPS markers linked to Rpi-blb2 (e.g. markers14L24L, 24Lspec, 24L9spec; FIG. 9).

Example 11 Physical Mapping of the S. bulbocastanum Derived Rpi-blb2Locus

The resistant clone Blb 2002 heterozygous for the Rpi-blb2 locus, wasused as source DNA for the construction of the S. bulbocastanum BAClibrary, hereafter referred to as the Blb 2002 BAC library. Highmolecular weight DNA preparation and BAC library construction werecarried out as described previously. A total of approximately 100.000clones were generated and stored as 50 bacterial pools containingapproximately 2000 white colonies. These bacterial pools were generatedby scraping the colonies from the agar plates into Luria Broth mediumcontaining 18% glycerol and 12.5 μg/ml chloramphenicol using a sterileglass spreader. For the screening of the Blb 2002 BAC library, plasmidDNA was isolated from each pool of clones using the standard alkalinelysis protocol and PCR was carried out to identify positive pools.Bacteria corresponding to positive pools were diluted and plated onLuria Broth agar plates containing chloramphenicol (12.5 μg/ml).Individual white colonies were subsequently picked into 384-wellmicrotiter plates and single positive BAC clones subsequently identifiedas described previously. Names of BAC clones isolated from the Blb 2002BAC library carry the prefix BlbSP.

In order to build a Blb 2002 derived BAC contig across the Rpi-blb2genetic marker interval (69L-24L) the Blb 2002 BAC library was screenedwith markers 141R and 24L. This lead to the isolation of BAC clonesBlbSP39 and BlbSP30, which overlap with each other and span the 141R-24Lmarker interval. BAC end sequences of both BAC clones were used todevelop the markers SPB30L and SPB39L (FIG. 9).

Example 12 Complementation Analyses

For complementation purposes, all Rpi-blb2 gene candidates, i.e. allMiGAs present on BAC clones BlbSP30, BlbSP39, 24, 242 and 211, weretargeted for subcloning into the binary vector pBINPLUS (van Engelen etal., 1996). This was done as follows. Aliquots of approximately 1 μg BACDNA were digested with 1 U, 0.1 U or 0.01 U of Sau3AI restriction enzymefor 30 min. The partially digested BAC DNA was subjected tocontour-clamped homogeneous electric field (CHEF) electrophoresis at 4°C. in 0.5×TBE using a linear increasing pulse time of 1-10 sec and afield strength of 6 V/cm for 16 hr. After electrophoresis, the agarosegel was stained with ethidium bromide to locate the region of the gelcontaining DNA fragments of approximately 10 kbp in size. This regionwas excised from the gel and treated with GELASE (EpicentreTechnologies, USA) according to the manufacturer. The size selected DNAwas ligated to the BamHI-digested and dephosphorylated binary vectorpBINPLUS (van Engelen et al., 1995) followed by transformation toElectroMAX E. coli DH10B competent cells (Life Technologies, UK). PerBAC clone a total of 384 clones were PCR screened for the presence ofMiGA sequences using the primers univ24L and univ14L (Table 3). Positiveclones were selected for further characterisation. Based on therestriction pattern of the 14L24L fragments digested with the enzymesRsaI, TaqI, AluI, DpnII or MseI, the different groups of MiGAs wereidentified. The MiGA harbouring the marker 24L, which was completelypresent on BAC clones BlbSP39, 211 and 242 was not detected with theuniversal primers univ14L and univ24L.

The relative position of the MiGA sequences in the 10 kbp subclones wasdetermined by PCR using internal primers 123Mi and 14L2 for the 5′ endand univ14L and 24L2 for the 3′ end in combination with primers derivedfrom pBINPLUS vector sequences (ARO 295 and 296; Table 3). Two subclonesper RGA of each BAC-library were selected for transformation.

For complementation analysis, the selected subclones were transferred tothe susceptible potato cultivars Impala and Kondor through Agrobacteriummediated transformation using isolate UIA143 (Farrand et al., 1989) orAGLO (Lazo et al., 1991). Primary transformants harbouring thetransgenes of interest were tested for resistance to P. infestans indetached leaf assays using isolate IPO655-2A and IPO82001 (Table 4).Only the genetic constructs harbouring RGC5, both derived from S.tuberosum and S. bulbocastanum, were able to complement the susceptiblephenotype both in cultivar Impala and in Kondor; in total 18 out of 19RGC5 containing primary transformants were resistant (Table 4, FIG. 12)whereas all RGC1, RGC2, RGC3, RGC4, RGC6 RGC7 or RGC8 genes containingprimary transformants were susceptible to P. infestans. As the RGC5transformants showed similar resistance phenotypes as the resistant S.bulbocastanum parent of mapping population B6, RGC5 was designated theRpi-blb2 gene. The homologue RGC24L can also be transferred to thedescribed susceptible potato cultivars and tested for resistance to P.infestans in a detached leaf assay.

A selection of primary transformants containing RGC5 was analysed forcopy number by Southern analysis. EcoRI digested genomic DNA washybridised with a nptII probe (Table 3). Based on the presence of thenumber of nptII hybridising fragments, the primary transformantscontained at least 1 to 11 transgene inserts. In total, 4 single copyintegrations in cultivar Impala and 6 in cultivar Kondor were observedof which one cultivar Kondor transformant appeared to have a P.infestans susceptible phenotype.

To investigate whether Rpi-blb2 can also complement the susceptiblephenotype in tomato, primary transformants of cultivar Moneymakerharbouring the Rpi-blb2 gene construct were produced and tested with thepotato derived isolates IPO82001 and IPO655-2A. The disease resistanceassay revealed that RGC5 is also able to complement a susceptible tomatophenotype (Table 6).

Example 13 Rpi-blb2 Gene Structure and Putative Amino Acid Sequence

The inserts of the RGC5 containing binary subclones 211FIC12 and SP39-20were sequenced by a primer walk strategy whereby consecutive rounds ofsequencing were carried out using a set of nested primers which weredesigned as the contiguous sequence was extended. The first set ofsequences was generated using the M13F and M13R primers. The completesequences of the inserts of clones 211FIC12 and SP39 consisted of 7967and 9949 nucleotides (nt), respectively (FIG. 13). The sequence of clone211F/C12 was identical to the corresponding sequence within cloneSP39-20. The position and putative structure of Rpi-blb2 was predictedusing GEN-SCAN (Burge and Karlin, 1997), GeneMark (Lukashin andBorodovsky 1998) and through alignment to the gene sequences of Mi1.1and Mi1.2.

The exact length and structure of the coding sequence was determinedthrough 5′ and 3′ rapid amplification of cDNA ends (RACE) using theGeneRacer™ kit (Invitrogen™ Groningen, the Netherlands). RACE identified5′ and 3′ Rpi-blb2 specific cDNA fragments comprising 5′ and 3′untranslated regions (UTRs) of 767 and 201 nucleotides (nt),respectively. The Rpi-blb2 gene contains two introns. Intron 1 is 626 ntlong and positioned within the 5′ UTR ending 32 nucleotides upstream ofthe ATG start codon. Intron 2 is 86 nt long starting 43 nucleotidesdownstream of the ATG start codon of the gene. The coding sequence ofthe Rpi-blb2 transcript is 3804 nucleotides.

The deduced open reading frame of the Rpi-blb2 gene encodes a predictedpolypeptide of 1267 amino acids with an estimated molecular weight of146 kD (FIG. 14). Several functional motifs present in R genes of theNBS-LRR class of plant R genes are apparent in the encoded protein. Asillustrated in FIG. 14, the Rpi-blb2 protein belongs to the leucinezipper (LZ) subset of NBS-LRR resistance proteins. The N-terminal halfof the Rpi-blb2 protein contains a potential LZ region between aminoacids 413 and 434 and six conserved motifs indicative of anucleotide-binding site (van der Biezen and Jones, 1998). The C-terminalhalf of Rpi-blb comprises a series of 15 irregular LRRs that can bealigued according to the consensus sequence hxxhxxLxxLxLxxC/N/SxLxxLPxx(SEQ ID NO: 100) or hxxhxxLxxLxLxxC/N/SxxLxxLPxx (SEQ ID NO: 101)observed in other cytoplasmic R proteins, whereby h can be L, I, M, V orF, and x any amino acid residue (Jones and Jones, 1997).

Example 14 Homology to Known State of the Art R Gene Sequences

To identify in silico homologues of the Rpi-blb2 gene, BLAST searches(Altschul et al., 1990) were carried out with the coding sequence of theRpi-blb2 gene. BLASTN searches identified a number of sequences withsignificant homology to the Rpi-blb2 gene. Using the alignment programmeClustalW (standard settings) in the DNAStar software package, wedetermined that the Rpi-blb2 coding sequence shares the highest homologyto Mi-1.1 (89.8%) and Mi-1.2 (89.7%) (Genbank accession numbers AF039681and AF039682, respectively). The latter sequence corresponds to the Migene from tomato that confers resistance to three of the most damagingspecies of the root knot nematodes (Meloidogyne spp.) (Milligan et al.,1998). In addition nucleotides 2410-3461 of the Rpi-blb2 coding sequenceshare 87.8% sequence homology to a partial NBS-LRR sequence from Solanumnigrum (Genbank accession number AY055116.1). At the amino acid levelthe putative Rpi-blb2 protein sequence shares the highest homology toMi-1.1 (82% identity) and Mi-1.2 (81% identity) (Genbank accessionnumbers AF039681 and AF039682).

Through ClustalW alignment of the deduced amino acid sequences ofRpi-blb2, Mi-1.1 and Mi-1.2 we have identified 200 amino acid (aa)residues which are unique to Rpi-blb2 (FIG. 15). Of these, 31 are foundat hypervariable positions, i.e. the residue at this position isdifferent in all three sequences and 11 are encoded by small insertions(one 3 aa residue insertion and one 8 aa residue insertion). The restare Rpi-blb2 specific in that the aa residues encountered atcorresponding positions in Mi-1.1 and Mi-1.2 are different from theRpi-blb2 residue but conserved in the two Mi protein sequences (FIG.15). Interestingly, the VLDL motif that is conserved in the third LRR ofmany NBS-LRR proteins including Mi (Axtell et al., 2001; Banerjee etal., 2001), is not conserved in Rpi-blb2 (FIG. 15).

Example 15 Rpi-blb2 Allele Mining in Wild Solanum Species

Using primers ARF1F and ARF1R (Table 3B), designed around the start andstop codon of the Rpi-blb2 gene, it is possible to amplify by PCR,alleles of Rpi-blb2 from any Solanum species. The amplification productscan be cloned between transcriptional regulatory sequences in a binaryplasmid and transferred to S. tuberosum through Agrobacterium mediatedtransformation or any method known to those skilled in the art. Theresulting primary transformants can subsequently be analysed forresistance to P. infestans or to any pathogen for which potato is a hostplant.

Example 16 Material and Methods

Plant material and development of mapping populations in (1) Solanumtuberosum. Complex interspecifc hybrid clones, designated ABPT, weremade by Hermsen and co-workers (Hermsen, 1966; Hermsen and Ramanna,1969; Ramanna and Hermsen, 1971; Hermsen and Ramanna, 1973; Hermsen,1983; Hermsen, 1994) (FIG. 1 a). The chromosome doubling step withcolchicines was described by Hermsen (1966) and Hermsen and De Boer(1971). The resistance in some of the ABPT clones to P. infestans isbelieved to be derived from either one or both of the accessions from S.bulbocastanum BGRC 8007 (CGN 21306; Pi 275196) and BGRC 8008 (CGN 17693;Pi 275198) that were used in the initial cross to produce hybridsbetween S. acaule and S. bulbocastanum, since all other parents thatwere used in the breeding scheme for ABPT-clones were susceptible oronly partially resistant to P. infestans in detached leaf assays(Hermsen and Ramanna, 1973). Tubers from 19 clones of population [(ABPTclone number 55× cultivar (cv) Oberarnbacher Frühe)×cv Arkula], from 7clones of population [(ABPT clone number 55×cv Oberarnbacher Frühe)×cvBlanka] and from 5 clones of population [(ABPT clone number 60×cvAlcmaria)×cv Blanka] were received in 1988 from the former Department ofPlant Breeding of the Wageningen Agricultural University (Wageningen,the Netherlands). Clones ARF 87-507, ARF 87-801 and ARF 87-601 wereselected from these populations respectively. They represented offspringfrom a second backcross (BC2) with the complex interspecific ABPT-clonesand were used for further back crosses that resulted in one tetraploïdBC3 population, two tetraploid BC4 populations and one diploid BC4population that were used for genetic mapping of the Rpi-blb2 gene (FIG.1). The tetraploid Solanum tuberosum mapping population ARG 95-15 wasproduced by crossing P. infestans resistant clone ARF 87-507 with thesusceptible cultivar Alkon. Tetraploid population ARG 95-3 was producedby crossing P. infestans resistant clone AR 91-1263 with the susceptiblecultivar Cosmos. Tetraploid population ARP 96-11 was produced bycrossing resistant clone AR 92-1292 with the susceptible cultivarCeleste. The diploid population DP1 was obtained by crossing theresistant clone ARD 1197-16 with the susceptible clone ARD 93-2090 (FIG.1).

Plant Material and Development of Mapping Populations in (2) Solanumbulbocastanum.

The diploid S. bulbocastanum mapping population, designated B6 (synonymB6a, Blb 99-229, Blb 00-7 and Blb 00-8), was developed by crossing a P.infestans resistant clone Blb 2002 (synonym M94-81-C) with a susceptibleclone Blb 48-5. Results from reciprocal crosses of population B6 werecombined. The resistant parental clone of population B6 was obtainedfrom a cross between S. bulbocastanum clone Blb 93-D26-3 (accession BGRC8002; CGN 17690; Pi 275187) as female parent and S. bulbocastanum cloneBlb 93-60-10 (accession BGRC 8006; Pi 275194) as male parent. Thesusceptible parental clone of population B6 was obtained from a crossbetween S. bulbocastanum clones from accessions BGRC 8005 (CGN 17692, PI275193) and BGRC 8006 (FIG. 2).

Disease Assays; (1) Phytophthora infestans Isolates

Three different P. infestans isolates were obtained from Plant ResearchInternational B. V. (Wageningen, the Netherlands). Isolates haddifferent race structures and mating types as follows: IPO82001: racestructure 1.2.3.4.5.6.7.10.11, mating type A2; IPO655-2A: race structure1.2.3.4.5.6.7.8.9.10.11, mating type A1; IPO428-2: race structure1.2.3.4.5.6.7.8.9.10.11, mating type A2 (Flier et al., 2003).

Disease Assays; (2) Field Trials

Glasshouse grown seedling tubers or field grown seed potatoes wereplanted at trial sites in Marknesse, the Netherlands from 1985 tot 2002,in the Toluca area of Mexico in 1991 or at a site in the BenguetProvince in the Philippines in 1992. For individual clones, plots wereplanted consisting of 1 to 10 tubers. Approximately 8 weeks afterplanting, the field at Marknesse was inoculated with a sporangiosporesolution of P. infestans isolate IPO82001 and disease scores werecollected 3 to 6 weeks after inoculation. Clones that were free ornearly free from late blight were classified as having a resistantphenotype whereas clones with a complete of nearly complete blightedfoliage were classified as susceptible. Clones with intermediatereactions to late blight were classified as having an unknown phenotype.At the field trials in Mexico and the Philippines, natural infection hadto occur. Once this natural infection by P. infestans established, thepercentage of blighted foliage of plants on each plot was scored on 8and 6 days respectively on a 1-9 scale were estimated percentages ofblighted foliage from 1 tot 9 were: 0, 3, 10, 25, 50, 75, 90, 97 and 100(Estrada-Ramos et al., 1983).

Disease Assays; (3) Detached Leaves

For the detached leaf assay, leaves from plants grown for 6 to 12 weeksin the greenhouse were placed in pieces of water-saturated floristsfoam, approximately 35×4×4 cm, and put in a tray (40 cm width, 60 cmlength and 6 cm height) with a perforated bottom. Each leaf wasinoculated with two droplets (25 μl each) of sporangiospore solution onthe abaxial side. Subsequently, the tray was placed in a plastic bag ontop of a tray, in which a water-saturated filter paper was placed, andincubated in a climate room at 17° C. and a 16 h/8 h day/nightphotoperiod with fluorescent light (Philips TLD50W/84HF and OSRAML58W/21-840). After 6 to 9 days, the leaves were evaluated for thedevelopment of P. infestans disease symptoms.

Evaluation:

Plants with leaves that clearly showed sporulating lesions 6 to 9 daysafter inoculation were considered to have a susceptible phenotype,whereas plants with leaves showing no visible symptoms or necrosis atthe side of inoculation in the absence of clear sporulation wereconsidered to be resistant.

Plant DNA Marker Screening

Genomic DNA was extracted from young leaves according to Bendahmane etal. (1997). For PCR analysis, 15 μl reaction mixtures were preparedcontaining 0.5 μg DNA, 15 ng of each primer, 0.2 mM of each dNTP, 0.6units Taq-polymerase (15 U/μl, SphaeroQ, Leiden, the Netherlands), 10 mMTris-HCl pH 9, 1.5 mM MgCl₂, 50 mM KCl, 0.1% Triton X-100 and 0.01%(w/v) gelatine. The PCRs were performed using the following cycleprofile: 25 seconds DNA denaturation at 94° C., 30 seconds annealing and40 seconds elongation at 72° C. As a first step in PCR-amplification DNAwas denatured for 5 min at 94° C. and finalised by an extra 5 minelongation step at 72° C. The amplification reactions were performed ina Biometra® T-Gradient or Biometra® Uno-II thermocycler (Westburg,Leusden, the Netherlands). Depending on the marker, the PCR product wasdigested with an appropriate restriction enzyme. An overview of themarkers including primer sequences, annealing temperature andrestriction enzymes if appropriate, is given in Table 3. Subsequently,the (cleaved) PCR products were analysed by electrophoresis in agaroseor acrylamide gels. For acrylamide gel analysis, the CleanGel DNAAnalysis Kit and DNA Silver Staining Kit (Amersham Pharmacia BiotechBenelux, Roosendaal, the Netherlands) were used.

Elongation of AFLP Fragments by Thermal Asymmetric Interfaced (TAIL)-PCR

Elongation of the sequence of an AFLP fragment was performed by TAIL-PCRaccording to Liu and Whittier (1995). Shortly, elongation of AFLPfragments was performed using 2 or 3 nested specific primers (sp) incombination with an arbitrary degenerate (AD) primer. The first PCR wasperformed with primers sp1 and AD, the second with sp2 and AD and thethird with sp3 and AD according to the scheme described in Table 5. ThePCR was performed in 25 μl reactions containing the standard PCR mix asdescribed before, except that 30 ng of primer AD was used. The elongatedfragments were cloned in pGEM-T (Promega, the Netherlands) andsequenced.

BAC Library Construction and Screening

The resistant clone ARD 1197-16, heterozygous for the Rpi-blb2 locus,was used as source DNA for the construction of the S. tuberosum BAClibrary. The resistant clone Blb 2002 heterozygous for the Rpi-blb2locus, was used as source DNA for the construction of the S.bulbocastanum BAC library. High molecular weight DNA preparation and BAClibrary construction were carried out as described in Rouppe van derVoort et al. (1999). For the S. tuberosum BAC library, approximately120.000 clones with an average insert size of 100 kb, which correspondsto 8 to 10 genome equivalents were finally obtained. A total ofapproximately 70.000 clones were individually stored in 177 384-wellmicrotiter plates at −80° C. Another 50.000 clones were stored as 14bacterial pools containing approximately 4000 white colonies. These weregenerated by scraping the colonies from the agar plates into Luria Brothmedium containing 18% glycerol and 12.5 μg/ml chloramphenicol using asterile glass spreader. These so-called super pools were also stored at−80° C. Finally, another 37.000 clones were added to the S. tuberosumBAC library. The S. bulbocastanum BAC library consisted of 48 superpools of approximately 2.000 colonies.

Marker screening of the BAC library harbouring the individually storedBAC clones was carried out as described in Rouppe van der Voort et al.(1999). For the screening of the BAC library stored as super pools,plasmid DNA was isolated from each pool of clones using the standardalkaline lysis protocol and PCR was carried out to identify positivepools. Bacteria corresponding to positive pools were diluted and platedon Luria Broth agar plates containing chloramphenicol (12.5 μg/ml)Individual white colonies were subsequently picked into 384-wellmicrotiter plates and single positive BAC clones subsequently identifiedas described above. Names of BAC clones isolated from the super poolscarry the prefix SP (e.g. SP39).

Subcloning of Candidate Genes

Candidate RGAs were subcloned from BAC clone 24, 211, 242, BLBSP39 andBLBSP30 as follows. Aliquots of approximately 1 μg BAC DNA were digestedwith 1 U, 0.1 U or 0.01 U of Sau3AI restriction enzyme for 30 min. Thepartially cleaved BAC DNA was subjected to CHEF electrophoresis at 4° C.in 0.5×TBE using a linear increasing pulse time of 1-10 sec and a fieldstrength of 6 V/cm for 16 hr. After electrophoresis, the agarose gel wasstained with ethidium bromide to locate the region of the gel containingDNA fragments of approximately 10 kbp in size. This region was excisedfrom the gel and treated with GELASE (Epicentre Technologies, USA)according to the manufacturer. The size selected DNA was ligated to theBamHI-cleaved and dephosphorylated binary vector pBINPLUS (van Engelenet al., 1995) followed by transformation to ElectroMAX E. coli DH10Bcompetent cells (Life Technologies, UK). A total of 192 clones were PCRscreened for the presence of RGC sequences using the primers of marker24L14L (Table 3). Positive clones were selected for furthercharacterisation. Identification of clones harbouring RGC1, RGC2, RGC3,RG4, RGC5, RGC6, RGC7, RGC8 and RGC24L was carried out by sequencing14L24L PCR fragments derived from positive clones. The relative positionof the RGAs within a subclone was determined by PCR analysis usinginternal primers (24L2, 123Mi) in combination with pBINPLUS specificprimers (Table 3).

Agrobacterium tumefaciens Mediated Transformation of Potato

Binary plasmids harbouring the candidate genes were transformed to A.tumefaciens strains AGL0 (Lazo et al., 1991) or UIA143 (Farrand et al.,1989), the latter containing the helper plasmid pCH32 (Hamilton et al.,1996). Overnight cultures of the transformed A. tumefaciens strains wereused to transform potato tuber discs (cvs Impala and Kondor) accordingto standard protocols (Hoekema et al., 1989; Fillati et al., 1987).Shortly, certified seed potatoes of cultivars Impala and Kondor werepeeled and surface sterilised for 30 min in a 1% sodium hypochloratesolution containing 0.1% Tween-20. Tubers were then washed thoroughly inlarge volumes of sterile distilled water (4 times, 10 min). Discs ofapproximately 2 mm thickness and 7 mm in diameter were sliced fromcylinders of tuber tissue prepared with a corkbore. The tuber discs weretransferred into liquid MS30 medium containing A. tumefaciens andincubated for 15 min. After removing the A. tumefaciens solution, thetuber discs were transferred to regeneration medium containing MS30, 0.9mg/l IAA, 3.6 mg/l zeatine riboside and 8 g/l agar (Hoekema et al.,1989). The plates were incubated at 24° C., 16 hour day-length (PhilipsTLD50W/84HF). After 48 hours of co-cultivation, the tuber discs wererinsed for 5 min in liquid MS medium including antibiotics, 200 mg/lvancomycin, 250 mg/l cefotaxim and 75 mg/l kanamycin, and transferred toregeneration medium supplemented with the same antibiotics. The plateswere incubated at 24° C., 16 hour day-length (Philips TLD50W/84HF).Every three weeks, the tuber discs were transferred to fresh medium.Regenerating shoots were transferred to MS30 medium containing 75 mg/lkanamycin. Rooting shoots were propagated in vitro and tested forabsence of A. tumefaciens cells by incubating a piece of stem in 3 mlLuria Broth medium (3 weeks, 37° C., 400 rpm). One plant of eachtransformed regenerant was transferred to the greenhouse.

Agrobacterium tumefaciens Mediated Transformation of Tomato

Seeds of the susceptible tomato line Moneymaker were rinsed in 70%ethanol to dissolve the seed coat and washed with sterile water.Subsequently, the seeds were surface-sterilised in 1.5% sodiumhypochlorite for 15 minutes, rinsed three times in sterile water andplaced in containers containing 140 ml MS medium pH 6.0 (Murashige andSkoog, 1962) supplemented with 10 g/l sucrose (MS10) and 160 mlvermiculite. The seeds were left to germinate for 8 days at 25° C. and0.5 W/M² light.

Eight day old cotyledon explants were pre-cultured for 24 hours in Petridishes containing a two week old feeder layer of tobacco suspensioncells plated on co-cultivation medium (MS30 pH 5.8 supplemented withNitsch vitamines (Duchefa Biochemie B V, Haarlem, the Netherlands), 0.5g/l MES buffer and 8 g/l Daichin agar).

Overnight cultures of A. tumefaciens were centrifuged and the pellet wasresuspended in cell suspension medium (MS30 pH 5.8 supplemented withNitsch vitamines, 0.5 g/l MES buffer, pH 5.8) containing 200 μMacetosyringone to a final O.D.₆₀₀ of 0.25. The explants were theninfected with the diluted overnight culture of A. tumefaciens UIA143containing pBINRGC5 for 25 minutes, blotted dry on sterile filter paperand co-cultured for 48 hours on the original feeder layer plates.Culture conditions were as described above.

Following the co-cultivation, the cotyledons explants were transferredto Petri dishes with selective shoot inducing medium (MS pH 5.8supplemented with 10 g/l glucose, including Nitsch vitamines, 0.5 g/lMES buffer, 5 g/l agargel, 2 mg/l zeatine riboside, 400 mg/lcarbenicilline, 100 mg/l kanamicine, 0.1 mg/l IAA) and cultured at 25°C. with 3-5 W/m² light. The explants were sub-cultured every 3 weeksonto fresh medium. Emerging shoots were dissected from the underlyingcallus and transferred to containers with selective root inducing medium(MS10 pH 5.8 supplemented with Nitsch vitamines, 0.5 g/l MES buffer, 5g/l agargel, 0.25 mg/l IBA, 200 mg/l carbenicillin and 100 mg/lkanamycine).

RNA Extraction

Total RNA was isolated using Trizol® according to the protocol suppliedby the manufacturer (Invitrogen™, Groningen, the Netherlands) with minormodifications. Briefly, 0.5 g of young leaf tissue was ground in liquidnitrogen and the powder suspended in 5 ml Trizol®. After a 5 minincubation at room temperature (RT), 0.5 ml chloroform was added, thesuspension was vortexed and incubated for 2 min. After centrifugation(15 min, 11404×g, 4° C.) the supernatant was transferred to a new tubeand 2.5 ml isopropanol was added. After 10 min at RT, nucleic acids wereprecipitated (10 min, 11404×g, 4° C.). The pellet was washed with 5 ml70% ethanol (5 min, RT) and after-centrifugation (5 min, 6415×g, 4° C.),the pellet was dried and resuspended in 100 μl sterile distilled water.PolyA+RNA was extracted from total RNA using the Oligotex™ mRNA midi kit(Qiagen, GmbH, Germany).

Rapid Amplification of cDNA Ends.

The 5′ and 3′ ends of the Rpi-blb2 cDNA and confirmation of putativeintron positions was determined by rapid amplification of cDNA ends(RACE) using the GeneRacer™ kit (Invitrogen™, Groningen, theNetherlands). 5′ RACE was carried out on cDNA synthesised with primerGSP4 (ARO 772). Subsequently, primer GSP6 (ARO 774) was used incombination with the GeneRacer™ 5′ primer and the final amplificationwas carried out with GSP6 in combination with the GeneRacer™ 5′ nestedprimer. 3′ RACE was carried out with the nested primers GSP1 (ARO 769)and GSP2 (ARO 770) in combination with the GeneRacer 3′ primer. Thefinal amplification was carried out with GSP3 (ARO 771) in combinationwith GeneRacer nested 3′ primer.

Both 3′ and 5′ RACE amplification steps were carried out using Accuprime(Invitrogen™, Groningen, the Netherlands) instead of the Taq polymerasesupplied by the GeneRacer™ kit.

AFLP Fingerprinting and Cloning of AFLP Fragments

Template preparation and AFLP fingerprinting were essentially performedas described in Vos et al. (1995). In order to clone specific fragments³³P-labelled AFLP fragments were excised out of the acrylamide gel byoverlaying the polyacrylamide gels, dried on Whatmann 3MM paper, withautoradiogram images. The pieces of gel/paper underneath the band ofinterest were cut out and transferred to 200 μl of TE and incubated for1 h at room temperature. Five microlitres of supernatant was used tore-amplify the fragment, using a PCR in which the EcoRI+0 in combinationwith MseI+0 were used as primers. The re-amplified AFLP fragment wassubsequently cloned into the pGEM-T cloning vector (Promega, theNetherlands) and the inserts of several clones sequenced.

The DNA sequence of the excised AFLP band was used to designlocus-specific primers. The amplification product obtained with suchprimers was screened for internal polymorphisms with restrictionenzymes. After restriction, the fragments were separated on a 2-3%agarose gel including ethidiumbromide.

RGA-AFLP Analysis

Template preparation was essentially performed as described in Vos etal. (1995). However, the second amplification step was carried out withthe P-loop based primer S1 from Leister et al. (1996) in combinationwith the EcoRI+0 AFLP primer. A 10 μl reaction mixture [0.5 μl³³P-labelled S1 primer (10 ng/μl); 0.5 μl EcoRI+0 primer (10 ng/μl); 0.8μl dNTPs (5 mM); 2 μl 10× Goldstar™ PCR buffer (Eurogenetc, Belgium);1.2 μl MgCl₂ (25 mM); 0.06 μl Goldstar™ DNA polymerase (5 U/μl)(Eurogentec, Belgium); 14.94 μl MQ water] was added to 10 μl dilutedtemplate (20× diluted in MQ water) and a PCR reaction performed usingthe following cycle profile: 45 seconds DNA denaturation at 94° C., 45seconds primer annealing at 49° C. and 2 min elongation step at 72° C.(35 cycles). Prior to the cycling the template DNA was denatured for 2min at 94° C. and the PCR was finalised by a applying an extra 5 minelongation step at 72° C. The amplification reactions were performed ina Perkin Elmer 9600 thermocycler. The labelled PCR products fragmentswere separated on a 6% polyacrylamide gel and the individual bandsvisualized by autoradiography according to standard procedures.

Example 17 Phenotype of Rpi-blb2 Expression

Material & Methods

Four lesions (6 days after inoculation at standard conditions) ofinfected leaflets (IPO82001) were rinsed in 3 ml H₂O. The concentrationwas determined using a haemocytometer Fuchs-Rosenthal (W. SchreckHofheim/Ts.)

Definition:

Sporulation index is the amount of sporangia per ml detected on lesionsof infected leaflets.

TABLE 7 Sporulation index of different genotypes after infection with P.infestans in a detached leaf assay Sporulation index Genotypesporangia/ml cv. Bintje 1.840.000 ARD 92-1197-16 20.000 R2-differential0

The difference between Rpi-blb2 and other P. infestans resistance genesis that Rpi-blb2 allows a low level of sporulation (FIG. 18). This isdemonstrated by a detached leaf assay in which the lesions present onRpi-blb2 genotype (ARD 92-1197-16) show a low level of sporangia inrelation to complete absence of sporangia on a genotype containing theS. demissum gene R2. However, the sporulation index is only 1.1% of asusceptible phenotype (cv. Bintje) (Table 7 and FIG. 18.

Field experiments have also shown that Rpi-blb2 allows a low level ofinfection. Late blight symptoms developed at a low level during thegrowing season (FIG. 3, ARF87-801) or at the end of the growing season(FIG. 2, ARF87-601; FIG. 3, ARF87-507 and ARF87-601).

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1. A method for generating or increasing the resistance of a plant to aplant pathogen of the phylum Oomyceta comprising increasing the activityof a Rpi-blb2 protein in the plant or a tissue, organ or cell of theplant or a part thereof by expressing a transgenic Rpi-blb2 proteinencoding nucleic acid molecule, wherein the plant has increasedresistance to the plant pathogen of the phylum Oomyceta, wherein saidRpi-blb2 protein encoding nucleic acid molecule is selected from thegroup consisting of: (a) a nucleic acid molecule encoding thepolypeptide depicted in SEQ ID NO: 2 or 4; (b) a nucleic acid moleculecomprising the coding sequence as depicted in SEQ ID NO: 3 or 5 or 6;and (c) a nucleic acid molecule encoding a polypeptide comprising asequence having at least 95% identity to the amino acid sequence of thepolypeptide encoded by the nucleic acid molecule of (a) or (b).
 2. Themethod of claim 1 which results in reduction in sporulation index of atleast 30% after infection with P. infestans compared to a wild type. 3.The method of claim 1, wherein the Rpi-blb2 protein comprises by aP-loop and a NBS domain.
 4. The method of claim 1, wherein the Rpi-blb2protein encoding nucleic acid molecule is a nucleic acid moleculeencoding a polypeptide comprising a sequence having at least 95%identity to the amino acid sequence of SEQ ID NO: 2 or 4, wherein thepolypeptide comprises a NBS domain and an LRR domain.